Overcoming problems with limiting DNA samples in forensics and clinical diagnostics using Multiple Displacement Amplification
Firman Alamsyah Muharam
Bachelor of Science / Bachelor of Arts, University of Queensland 2002
Cooperative Research Centre for Diagnostics School of Life Science, Faculty of Science Queensland University of Technology Brisbane, Australia
A thesis submitted for the degree of Master of Applied Science (Research) at the Queensland University of Technology, Brisbane, Australia 2005
i
ii ABSTRACT
The availability of DNA samples that are of adequate quality and quantity is essential for any genetic analysis. The fields of forensic biology and clinical diagnostic pathology testing often suffer from limited samples that yield insufficient DNA material to allow extensive analysis. This study examined the utility of a recently introduced whole genome amplification method termed
Multiple Displacement Amplification (MDA) for amplifying a variety of limited sample types that are commonly encountered in the fields of forensic biology and clinical diagnostics. The MDA reaction, which employs the highly processive bacteriophage φ29 DNA polymerase, was found to generate high molecular weight template DNA suitable for a variety of downstream applications from low copy number DNA samples down to the single genome level. MDA of single cells yielded sufficient DNA for up to 20,000,000 PCR assays, allowing further confirmatory testing on samples of limited quantities or the archiving of precious DNA material for future work. The amplification of degraded DNA material using MDA identified a requirement for samples of sufficient quality to allow successful synthesis of product DNA templates.
Furthermore, the utility of MDA products in comparative genomic hybridisation
(CGH) assays identified the presence of amplification bias. However, this bias was overcome by introducing a novel modification to the MDA protocol. Future directions for this work include investigations into the utility of MDA products in short tandem repeat (STR) assays for human identifications and application of the modified MDA protocol for testing of single cell samples for genetic abnormalities.
iii KEYWORDS
Whole genome amplification, multiple displacement amplification, φ29, phi29,
DNA polymerase, single cell, low copy number, DNA, PCR, forensic biology, forensic science, clinical diagnostics, comparative genomic hybridisation.
iv TABLE OF CONTENTS
ABSTRACT ...... iii KEYWORDS...... iv TABLE OF CONTENTS...... v LIST OF FIGURES...... vii LIST OF TABLES ...... viii LIST OF ABBREVIATIONS AND SYMBOLS...... ix LIST OF PRESENTATIONS AND PUBLICATIONS ...... xi STATEMENT OF ORIGINALITY ...... xii ACKNOWLEDGEMENTS...... xiii
CHAPTER ONE: AN INTRODUCTION TO THE PROBLEMS OF LIMITING DNA SAMPLES AND THE AIMS OF THIS RESEARCH: WHOLE GENOME AMPLIFICATION IN FORENSICS AND CLINICAL DIAGNOSTICS ...... 1 1.1. Introduction ...... 1 1.2. The Problem of Limiting DNA Samples...... 2 1.2.1. The need for large amounts of DNA for high-throughput genetic studies ...... 2 1.2.2. The amount of DNA in single cells and examples of limiting samples...... 4 1.2.2.1. Limiting samples in the field of forensic DNA testing ...... 5 1.2.2.2. Limiting samples in the field of molecular archaeology ...... 10 1.2.2.3. Limiting samples in the field of clinical diagnostics ...... 12 1.2.2.4. Overcoming the problem of limited samples...... 15 1.3. Applying Whole Genome Amplification (WGA) Methods to Overcome Problems with Limiting Sample Quantities...... 16 1.3.1. PCR-based Whole Genome Amplification ...... 18 1.3.1.1. Alu-PCR, Linker Adapter PCR (LA-PCR) and Tagged Random PCR (T-PCR)...... 20 1.3.1.2. Degenerate oligonucleotide-primed PCR (DOP-PCR)...... 23 1.3.1.3. Primer Extension Preamplification (PEP)...... 28 1.3.1.4. Summary on the characteristics of PCR-based WGA methods...... 31 1.3.2. Non-PCR-based isothermal Whole Genome Amplification by strand- displacing Bst and φ29 DNA polymerases...... 33 1.3.2.1. Rolling Circle Amplification (RCA) by Large Fragment Bst DNA polymerase ...... 34 1.3.2.2. Multiple Displacement Amplification (MDA) by φ29 DNA polymerase ...... 37 1.4. Research Rationale: Background to the Investigation...... 46 1.5. Hypothesis and Research Aims...... 48 1.5.1. Hypothesis ...... 48 1.5.2. Research Aim I ...... 49 1.5.3. Research Aim II...... 49 1.5.4. Research Aim III ...... 50 1.6. Research Design...... 50 1.7. Thesis Outline ...... 50 1.8. Conclusion...... 53
CHAPTER TWO: MATERIALS AND METHODS ...... 54 2.1. Introduction ...... 54 2.2. Sample Handling and Preparation of DNA Samples...... 54 2.2.1. General sample handling ...... 54 2.2.2. Dilution of human genomic DNA for sensitivity assays ...... 56 2.2.3. Collection of human fibroblasts for sensitivity assays ...... 56 2.2.4. Collection and preparation of simulated forensic specimens (buccal swabs, licked stamps, touched object samples and shed hairs) ...... 57 2.3. DNA Extraction and Quantification ...... 58 2.3.1. DNA extraction of population database bloodspots by 5% Chelex-100 ...... 56 2.3.2. DNA extraction of simulated low copy number forensic samples (buccal swabs, licked stamps, touched object samples) by a modified 5% Chelex protocol...... 59
v 2.3.3. Extraction of DNA from shed hairs by a modified Buffer/Proteinase K digest protocol...... 59 2.3.4. Procedure for salt extraction of genomic DNA from whole blood for use in Comparative Genomic Hybridisation (CGH) ...... 60 2.3.5. Quantification of samples by quantitative real-time PCR (RT-QPCR) ...... 61 2.3.6. Quantification of whole blood genomic DNA and MDA products by spectrophotometric 260nm/280nm absorbance ...... 62 2.4. PCR Amplification and Agarose Gel Electrophoresis ...... 63 2.5. Multiple Displacement Amplification (MDA) ...... 66 2.5.1. Multiple Displacement Amplification (MDA) using the GenomiPhi™ DNA Amplification Kit ...... 66 2.5.2. A Modified MDA procedure using the GenomiPhi™ DNA Amplification Kit...... 67 2.6. Artificial Degradation of Genomic DNA by Bovine Pancreatic DNase I ...... 68 2.7. Comparative Genomic Hybridisation (CGH) ...... 69 2.8. Conclusion...... 71
CHAPTER THREE: APPLICATION OF MULTIPLE DISPLACEMENT AMPLIFICATION IN FORENSIC BIOLOGY ...... 72 3.1. Introduction ...... 72 3.2. Results and Discussion ...... 78 3.2.1. Sensitivity and yield of MDA on low DNA concentrations down to one single human cell...... 78 3.2.2. MDA of Simulated Forensic Samples...... 84 3.2.3. The Effect of Degraded DNA Templates on GenomiPhi™ MDA...... 91 3.2.4. Archiving of precious population samples...... 96 3.3. Summary and Conclusions...... 102 3.3.1. Guidelines for amplifying limited forensic DNA samples by MDA using the GenomiPhi™ DNA Amplification Kit (Amersham Biosciences, Piscataway, NJ, USA)...... 107 3.3.2. Guidelines for the utility of MDA for archiving fresh bloodspot samples, using the GenomiPhi™DNA Amplification kit (Amersham Biosciences, Piscataway, NJ, USA) ...... 111
CHAPTER FOUR: APPLICATION OF MULTIPLE DISPLACEMENT AMPLIFICATION IN CLINICAL DIAGNOSTICS...... 113 4.1. Introduction ...... 113 4.2. Results and Discussion ...... 124 4.2.1. Assessment on the possibility of misdiagnosis caused by MDA products from a negative (no DNA) reaction control ...... 126 4.2.2. Amplification bias in MDA-amplified normal human genomic DNA ...... 128 4.3. Summary and Conclusions...... 145
CHAPTER FIVE: FINAL SUMMARY AND CONCLUSIONS ...... 148
REFERENCES...... 156
vi LIST OF FIGURES
Figure 1.1. Examples of limiting sample types that are analysed in the fields of forensics or clinical diagnostics...... 5 Figure 1.2. Simplified schematic of the comparative genomic hybridisation (CGH) protocol...... 13 Figure 1.3. A schematic of the multiple displacement amplification (MDA) reaction...... 39 Figure 3.1. Agarose gel analysis of MDA and PCR reactions from genomic DNA or single cell dilutions...... 79 Figure 3.2. Agarose gel analysis of product from the PCR amplification of DNA template from a two-cell MDA reaction diluted by 10-fold serial dilution to 1:1,000,000...... 82 Figure 3.3. Amplification fold and yield after MDA of simulated forensic samples...... 89 Figure 3.4. Comparison of successful single-locus PCR of swab samples, with and without MDA...... 89 Figure 3.5. Agarose gel analysis of artificially-degraded genomic DNA...... 92 Figure 3.6. Artificially degraded DNA fragments after amplification by MDA...... 93 Figure 3.7. PCR analysis of the MDA reactions shown in Figure 3.6...... 94 Figure 3.8. Inhibition of bloodspot samples...... 101 Figure 3.9. Downstream PCR amplification of MDA reaction products from diluted bloodspot extracts...... 101 Figure 4.1. A diagram depicting representations of all 23 pairs of human chromosomes and the CGH profile of a patient DNA sample...... 116 Figure 4.2. An illustration depicting the process of preimplantation genetic diagnosis (PGD)...... 121 Figure 4.3. CGH profile for a normal human male genomic DNA sample...... 125 Figure 4.4. CGH analysis of non-specific MDA products...... 127 Figure 4.5. CGH profiles for MDA-amplified normal human male genomic DNA (a) with Cot1 suppression and (b) without Cot1 suppression for repetitive human DNA sequences...... 130 Figure 4.6. Non-random distribution of amplification bias...... 132 Figure 4.7. Chromosomal regions affected by MDA amplification bias...... 132 Figure 4.8. Correlation between a region of MDA over-amplification on chromosome 6 to a region containing a high concentration of known genes...... 134 Figure 4.9. Correlation between other regions of MDA amplification bias to locations of high or low gene density...... 135 Figure 4.10. CGH profiles for MDA reactions where the denaturation step (95°C for 3 minutes) for denaturing sample DNA was either (a) included or (b) omitted...... 140 Figure 4.11. Methods used for mixing denatured and non-denatured MDA products or DNA templates...... 142 Figure 4.12. CGH profiles from MDA products that were generated using modified GenomiPhi™ method I or II...... 143
vii LIST OF TABLES
Table 1.1. The 13 core STR loci used for the Combined DNA Index System (CODIS) DNA database in the USA...... 6 Table 1.2. Historical or scientific relevance of recent findings within the field of molecular archaeology, where ancient DNA sources were used as the primary research material...... 11 Table 2.1. Primer sequences for quantitative real-time PCR amplification on the ABI 7000 SDS platform...... 62 Table 2.2. Nuclear and mitochondrial DNA targets and thermocycling conditions used for PCR amplification...... 65 Table 3.1. Amplification fold and final yield for single cell samples amplified by MDA...... 81 Table 3.2. Assessment of simplified extraction protocols for use with forensic samples...... 87 Table 3.3. Amplification fold and maximum yield for various types of simulated forensic specimens from MDA of 1µL extracted DNA...... 88 Table 3.4. Fragment size ranges of degraded DNA, artificially fragmented by a dilution of bovine pancreatic DNase I enzyme...... 91 Table 3.5. Results for PCR of artificially degraded fragments pre- and post- MDA using short (approximately 100bp) PCR primer sets...... 94 Table 3.6. PCR amplification of extracted ethnic population samples...... 98 Table 3.7. Potential total yield from MDA of diluted fresh bloodspot extracts (in number of single locus PCR reactions)...... 102 Table 4.1. Major disorders of the autosomes and sex chromosomes (adapted from Nussbaum et al., 2001)...... 117
viii LIST OF ABBREVIATIONS AND SYMBOLS
°C degrees Celcius φ phi µg micrograms µL microliters µM micromolar A adenine aDNA ancient DNA BAC bacterial artificial chromosomes bp base pairs BSA bovine serum albumin cDNA complementary DNA C cytosine CGH comparative genomic hybridisation CVS chorionic villus sampling DAPI 4’,6-diamidino-2-phenylindole dATP 2’-deoxyadenosine 5’-triphosphate dCTP 2’-deoxycytidine 5’-triphosphate dGTP 2’-deoxyguanosine 5’-triphosphate DMSO dimethyl sulfoxide DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphates DOP-PCR degenerate oligonucleotide-primed polymerase chain reaction dsDNA double-stranded deoxyribonucleic acid dTTP 2’-deoxythimidine 5’-triphosphate dUTP 2’-deoxyuridine 5’-triphosphate DVI disaster victim identification EDTA ethylenediamine tetraacetic acid EGTA ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid ET buffer ethylenediamine tetraacetic acid tris-hydrochloric acid buffer fg femtogram FISH fluorescence in situ hybridisation G guanine gDNA genomic deoxyribonucleic acid GEq genomic equivalents HCl hydrochloric acid hr hours I-PEP improved primer extension preamplification ICSI intracytoplasmic sperm injection IVF in vitro fertilisation kb kilobase pairs kDa kilo Dalton LA-PCR linker adapter polymerase chain reaction, also see LMP LL-DOP-PCR long products from low DNA quantities DOP-PCR LMP ligation mediated polymerase chain reaction MDA multiple displacement amplification mg milligrams min minutes mL milliliters mmol millimoles mtDNA mitochondrial deoxyribonucleic acid ng nanograms ng/µL nanograms per microliter nm nanometer nuDNA nuclear deoxyribonucleic acid p chromosome short arm PBS phosphate-buffered saline PCR polymerase chain reaction
ix PEP primer extension preamplification pers.comm. personal communication pg picograms pg/µL picograms per microliter PGD preimplantation genetic diagnosis pH pondus hydrogenii, potency of hydrogen (H+) ions pmol picomoles PRSG adapter-ligation PCR of randomly sheared genomic DNA q chromosome long arm RCA rolling circle amplification RFLP restriction fragment length polymorphism SCOMP single cell comparative genomic hybridisation SDA strand displacement activity SDS sodium dodecyl sulfate sec seconds SNP single nucleotide polymorphism STR short tandem repeat T thymine TAE buffer tris-acetate-EDTA buffer TBE buffer tris-borate-EDTA buffer TE buffer tris-hydrochloric acid ethylenediamine tetraacetic acid buffer T-PCR tagged random polymerase chain reaction U units UDG uracil DNA-glycosylase UV ultraviolet light WCP whole chromosome painting WGA whole genome amplification
x LIST OF PRESENTATIONS AND PUBLICATIONS
1. Muharam F.A., Hodgson R.A., van Daal A. (2004). 2,000,000 PCR reactions generated by whole genome amplification of limited forensic samples. The 7th International Conference on Ancient DNA and Associated Biomolecules (DNA7). Brisbane, Queensland, AUSTRALIA, 10-17 July 2004 (poster presentation).
2. Muharam F.A., Hodgson R.A., Westacott L., White S., van Daal A. (2004). Forensic Applications of Whole Genome Amplification. The 7th International Conference on Ancient DNA and Associated Biomolecules (DNA7). Brisbane, Queensland, AUSTRALIA, 10-17 July 2004 (oral presentation by A. van Daal).
3. Muharam F.A., Hodgson R.A., van Daal A. (2004). Two million PCR reactions generated from difficult forensic samples. 15th International Symposium on Human Identification. Phoenix, Arizona, UNITED STATES OF AMERICA, 4-7 October 2004 (poster presentation).
4. Muharam F.A., Hodgson R.A., Westacott L., White S., van Daal A. (2004). Forensic Applications of Whole Genome Amplification. 15th International Symposium on Human Identification. Phoenix, Arizona, UNITED STATES OF AMERICA, 4-7 October 2004 (oral presentation by A. van Daal).
5. Muharam F.A., Hodgson R.A., van Daal A. (2005). Overcoming problems with limiting DNA samples: Multiple Displacement Amplification in Forensics (submitted as a Technical Note to the Journal of Forensic Sciences on 14 July 2005).
xi STATEMENT OF ORIGINALITY
The research presented in this thesis is my original work (except where otherwise acknowledged). It was completed in September 2005 and submitted in fulfilment of a Master of Applied Science (Research) in the School of Life Science, Faculty of Science, at the Queensland University of Technology (Brisbane, Queensland, Australia). The work contained in this thesis has not been previously submitted for a degree or diploma at any other higher education institution.
F.A. Iman Muharam BSc/BA 24 September 2005 Brisbane, Queensland, Australia
xii ACKNOWLEDGEMENTS
I would like to thank my supervisors Assoc. Prof. Angela van Daal, Dr Richard Hodgson and Assoc. Prof. Phillip Morris for giving me the opportunity to undertake this project. Their support has helped me throughout the duration of my candidature at QUT, which was only supposed to be two years but extended to three! Angela and Richard were always available for consulting and trouble- shooting when experiments did not produce the expected outcomes. It was sad to see Richard leave in early 2004 for employment elsewhere, but he still made himself available for technical-related questions. I must thank all of my supervisors who provided me with opportunities and support to attend seminars and conferences both nationally and internationally, as well as introducing me to other researchers and scientists in my related fields. I must also thank Angela for helping me with the selection criteria for my first science-related job: a “CSI” at Queensland Health!
I thank Assoc. Prof. Phillip Morris, Dr Ron Epping and other staff from the Cooperative Research Centre for Diagnostics, and also the School of Life Science, who provided me with funding for conference travel to Phoenix, Arizona (USA) in 2004, which was attended by more than 500 forensic scientists from all over the globe. It was a very fruitful journey that helped me to become a more professional and confident scientist, and helped increase my contacts in the world of forensic science.
I must thank members of staff from other institutions who have also helped me throughout the project. Thanks go to Ms Sue White, Dr Lorraine Westacott and Mrs Pam Tinniswood from the Genetics Department, Queensland Medical Laboratory (QML) at West End, Brisbane, for allowing me to complete part of my project in their lab. Pam was a great mentor in comparative genomic hybridisation, and was extremely patient when things weren’t working (eg. poor hybridisation, messy backgrounds and the UV lamp going bust!). I owe Pam for all the hours she spent in front of the computer, karyotyping my CGH results. I also thank staff from the Archaeological Sciences Laboratory at the University of Queensland, especially Dr Tom Loy and Ken Dusza, for assisting me with ancient DNA extraction procedures. MATP and mitochondrial DNA primers
xiii were gifts from Justin Graf and Sol York (CRC for Diagnostics, QUT) respectively.
Thanks go to fellow QUT CRC Dx students who helped keep me sane throughout my time there. I also thank my workmates at Forensic Biology, Queensland Health, for constantly enquiring about the status of my thesis and telling me to quickly finish writing up, and giving me many days off to do this! I must thank Apple Computer Inc. for designing great computers (eg. iMac G5!) that don’t crash every 10 minutes.
Last but not least, I thank my family for all their guidance and support for the past three years, especially for keeping the noise down whenever I’m writing. I can’t thank you enough.
“Can’t sing but I’ve got soul, the goal is elevation”
U2, ELEVATION
(ALL THAT YOU CAN’T LEAVE BEHIND, 2000)
Iman Muharam September 2005 Brisbane, Queensland, Australia
xiv CHAPTER ONE
AN INTRODUCTION TO THE PROBLEMS OF
LIMITING DNA SAMPLES AND THE AIMS OF THIS
RESEARCH: WHOLE GENOME AMPLIFICATION IN
FORENSICS AND CLINICAL DIAGNOSTICS
1.1. Introduction
This thesis is concerned with the use of whole genome amplification
(WGA) for amplifying DNA samples of limited quantities (referred to as
“limited or limiting DNA samples” throughout this thesis) as encountered in the disciplines of forensic biology and clinical molecular diagnostics. DNA amplification technologies such as the polymerase chain reaction (PCR) or WGA involve the production of DNA to amounts suitable for analysis. DNA amplification technologies may involve a technique for the specific amplification of a particular region of interest, as performed in the polymerase chain reaction (PCR), or the non-specific amplification of all DNA from samples containing low amounts of DNA in order to increase the amount of template DNA for
1 extensive genetic analysis, as performed by WGA. The ability to successfully amplify low amounts of sample DNA is a prerequisite for many molecular biology assays in several diverse fields such as forensic biology, molecular archaeology and clinical diagnostics. DNA amplification technologies are thus a valuable tool for any DNA laboratory.
In this chapter, I present an overview of the literature on current approaches to overcome the problems associated with the use of low copy DNA samples, followed by my research aims and the research design. A summary of each following chapter is also presented.
1.2. The Problem of Limiting DNA Samples
1.2.1. The need for large amounts of DNA for high-throughput genetic studies
At the most basic level, the isolation of sufficient quantity and quality of
DNA is a fundamental requirement for any molecular biology analysis.
However, this prerequisite can often become an obstacle for many DNA testing applications. Prior to the 1980’s, the field of molecular biology was limited by the availability of sample DNA as microgram quantities were required for testing, whereas only 6pg of DNA is available from
2 any one human diploid cell (Dolezel et al., 2003). As any particular sample of interest may only consist of a few hundred cells, the DNA yield is often insufficient for extensive analysis. The description of the polymerase chain reaction (PCR) in the mid 1980’s (Saiki et al., 1985;
Mullis and Faloona, 1987) was therefore hailed as a major scientific breakthrough as it became possible to amplify sufficient quantities of specific regions of DNA. PCR technology has since become an indispensable tool for any laboratory performing genetic analysis that requires large amounts of DNA.
The completion of the human genome sequencing effort has triggered an increase in the availability of molecular biology tools and technologies for interrogating the information contained within our DNA, such as complex physical traits or disease heritability genetics that may assist clinicians in identifying individuals at risk of genetic disorders prior to manifestation of a disease phenotype. These new discoveries and developments allow scientists and researchers to extract more genetic information from any one particular sample by adapting high-throughput techniques.
However, high-throughput genetic analysis requires large amounts of template for testing. A genome-wide single-nucleotide polymorphism
(SNP) assay for the detection of a disease-causing gene may investigate up to 500,000 SNP’s and require up to 5mg of DNA if 10ng of sample is sufficient per SNP assay (Grant et al., 2002). Amplifying individual
3 target regions in limiting samples by conventional PCR is reliable but multiplex reactions and increased throughput may be necessary in order to maximise data obtained from limiting samples. However, multiplex
PCR design is difficult and only with very precise optimisation is a 50- to 60-plex possible (Wang et al., 1998). Furthermore, DNA analysis methods, especially high-throughput genetic studies, are constantly becoming more demanding in their need for robust and representative
DNA samples of substantial quality and quantity to enable many downstream analyses. Conventional PCR alone does not meet these requirements, and in combination with the above factors may limit the type and number of high-throughput assays that can be performed.
1.2.2. The amount of DNA in single cells and examples of limiting samples
Estimates for the genome sizes of single human female and male diploid cells are 6.406x109 and 6.294x109 bases respectively (International
Human Genome Sequencing Consortium, 2001; Dolezel et al., 2003), the female genome being larger due to the size difference between the X and Y sex chromosomes. Consequently, a single diploid human female or male cell nucleus in the G1 phase contains 6.550 and 6.436pg of DNA respectively (Dolezel et al., 2003). One milligram of sample may therefore contain up to 1.56x108 cells. However, more realistic sample sizes that are encountered in forensics and clinical diagnostics are in the sub-nanogram range. Several limiting sample types that are often
4 analysed in the fields of forensics and diagnostics are illustrated in
Figure 1.1. Of note, the analysis of single cells such as spermatozoa or oocytes often occurs in the field of clinical diagnostics. In these cases, the single cell can only be analysed once, and confirmatory testing of any one cell is often difficult to accomplish.
Figure 1.1. Examples of limiting sample types that are analysed in the fields of forensics or clinical diagnostics. From top, left to right: saliva stains on cigarette butts, licked stamps, latent fingerprints, archaeological mummified tissue, single blastomeres, spermatozoa, blood stains and single shed hair roots. The nucleus of a human diploid cell only contains up to 6.55pg of genomic DNA, and the number of viable cells available for analysis from these samples is often very limited.
1.2.2.1. Limiting samples in the field of forensic DNA testing
It is widely accepted that the typical DNA yield from individual samples can be a limiting factor, especially in areas where test samples only contain trace amounts of DNA as encountered in single-cell diagnostics, forensic biology, and molecular archaeology. There is an increasing demand specifically in the field of forensic biology to analyse every piece of evidence for trace DNA deposits that may aid a criminal
5 investigation. Forensic DNA typing utilises microsatellite DNA regions with repeat units that are 4bp in length, termed tetranucleotide short tandem repeats (STR). The use of STR markers has become popular because they are easily amplified by PCR in multiplex reactions where numerous STR markers are co-amplified simultaneously, generating
PCR fragments between 97-464bp in length (Butler, 2005), thus enabling the analysis of moderately degraded DNA samples.
Furthermore, the number of 4bp repeats in each STR marker is highly variable between individuals, and is therefore an effective tool for human identification purposes.
Table 1.1. The 13 core STR loci used for the Combined DNA Index System (CODIS) DNA database in the USA.
STR Locus Chromosomal Repeat Motif Repeat Name Location Range* CSF1PO 5q33.1 TAGA 5-16 FGA 4q31.3 CTTT 12.2-51.2 TH01 11p15.5 TCAT 3-14 TPOX 2p25.3 GAAT 4-16 vWA 12p13.31 [TCTG][TCTA] 10-25 D3S1358 3p21.31 [TCTG][TCTA] 8-21 D5S818 5q23.2 AGAT 7-18 D7S820 7q21.11 GATA 5-16 D8S1179 8q24.13 [TCTA][TCTG] 7-20 D13S317 13q31.1 TATC 5-16 D16S539 16q24.1 GATA 5-16 D18S51 18q21.33 AGAA 7-39.2 D21S11 21q21.1 Complex 12-41.2 [TCTA][TCTG] * Number of repeat units present in the alleles. Data adapted from STRbase, http://www.cstl.nist.gov/div831/strbase/ (Ruitberg et al., 2001).
6 In 1997, a project team led by the Federal Bureau of Investigation (FBI) chose 13 core STR loci, plus Amelogenin for sex identification, to be the basis of the future Combined DNA Index System (CODIS) national
DNA database in the USA (Budowle et al., 1998) (refer to Table 1.1).
Forensic scientists worldwide routinely utilise combinations of these
STR loci, commercially available as ready-made kits such as
PowerPlex® 16 (Promega Corp., Madison, WI, USA) and AmpFlSTR®
Profiler Plus® (Applied Biosystems, Foster City, CA, USA), to aid criminal investigations and human identifications. For a DNA typing test where the combination of all 13 CODIS STR loci are analysed, the average random match probability is rarer than one in a trillion among unrelated individuals (Chakraborty et al., 1999).
Current forensic DNA testing by short tandem repeat (STR) PCR requires approximately 1 to 2.5ng of DNA to ensure optimum amplification with the widely used AmpFlSTR® Profiler Plus® kit from
Applied Biosystems (Applied Biosystems, 1998), a major manufacturer of forensic identity testing products. Presently there are still many samples collected from crime scenes that do not contain sufficient DNA material for successful STR analysis. Examples of forensic exhibits that may contain limited amounts of DNA include blood stains (Budowle et al., 1995); semen stains (Budowle et al., 1995); bones (Gill et al., 1994); teeth (Alvarez Garcia et al., 1996); hair roots (Higuchi et al., 1988) and hair shafts (Wilson et al., 1995); saliva stains (Sweet et al., 1997) such as those on cigarette butts (Hochmeister et al., 1991) and licked postage
7 stamps (Hopkins et al., 1994) or envelope sealing flaps (Word and
Gregory, 1997); urine (Benecke et al., 1996); faeces (Hopwood et al.,
1996); fingernail scrapings (Wiegand et al., 1993); dandruff (Herber and
Herold, 1998); touched objects or latent fingerprints (van Oorschot and
Jones, 1997); and various personal items, such as chewing gum, toothbrush and ear wax (Tahir et al., 1996).
For the purposes of forensic DNA typing, it is important to extract the
DNA using methods that avoid further degradation of the DNA material and are able to remove inhibitors that can affect downstream PCR amplification assays. Inhibitors present in forensic samples can (1) interfere with cell lysis during DNA extraction by inhibiting the activity of proteolytic enzymes such as Proteinase K, (2) interfere by nucleic acid degradation or capture and (3) inhibit DNA polymerase activity during PCR (Wilson, 1997). Examples of well-known inhibitors often encountered in forensic samples include the textile dye in blue jeans
(Shutler et al., 1999), heme in blood (Akane et al., 1994), melanin in tissue and hair (Eckhart et al., 2000), and humic compounds in soil (Tsai and Olson, 1992). The effect of amplifying a sample containing an excess of inhibitors is a loss of the larger sized STR loci or complete failure of the PCR reaction. However, diluting the DNA sample (and therefore co-diluting any inhibitors present) prior to PCR can often overcome the effect of inhibition. Alternatively, additives such as bovine serum albumin (BSA) (Comey et al., 1994) or betaine (Al-Soud and
Rådström, 2000) can help minimise the inhibition of PCR. Further
8 purification of the DNA extracts to remove inhibiting compounds can also be performed using devices such as the Centricon-100 or Microcon-
100 filters (Comey et al., 1994).
DNA degradation is more difficult to overcome than excess amounts of inhibitors. DNA degradation occurs via both chemical and enzymatic mechanisms (Lindahl, 1993). After the death of a cell or organism, DNA molecules are rapidly subjected to degradation by cellular nucleases, followed by bacteria, fungi and insect infestation (Poinar, 2003). Any remaining DNA is then subject to hydrolytic cleavage and oxidative base damage. Hydrolytic cleavage primarily targets the glycosidic base sugar bond, resulting in base loss through depurination and subsequent nicking of the DNA (Poinar, 2003). The rate of hydrolytic cleavage is increased with heat and humidity, causing direct strand cleavage of the DNA due to drying (Poinar, 2003). DNA degradation can also be caused by oxidative damage, whereby oxidation of carbon bonds in pyrimidines and imidazole rings in purines results in ring fragmentation (Lindahl,
1993). Fragmentation of DNA molecules at regions where primers anneal or bind, or in regions internal to the primers, can significantly reduce the efficiency and success of the PCR amplification.
Furthermore, oxidised base products are not by-passed with standard
Taq DNA polymerase as used in PCR (Evans et al., 1993), and UV irradiation of DNA can lead to cross-linking of adjacent thymine nucleotides, preventing passage of the DNA polymerase during PCR
(Butler, 2005).
9 1.2.2.2. Limiting samples in the field of molecular archaeology
The problem of limited DNA sample is also common in the field of molecular archaeology, the study of ancient DNA. Ancient DNA (also commonly referred to as aDNA) was first analysed in the 1980’s with the introduction of the PCR protocol (Pääbo, 1985; Pääbo et al., 1989;
Pääbo, 1989). Molecular archaeology evolved from attempts to characterise DNA material from archaeological samples and has allowed the genetics of ancient or extinct species to be studied in relation to present species (Loy, 1983; Loy and Matthaei, 1994; Hofreiter et al.,
2001), as well as the investigation of the phylogenetic relationship between members of the genus Homo (Stringer and Andrews, 1988;
Krings et al., 1997; Krings et al., 1999; Krings et al., 2000), and characterisation of interactions between ancient humans and their surroundings (Loy, 1988; Loy and Hardy, 1992; Loy and Dixon, 1998)
(Table 1.2). Ancient DNA studies have also identified the impact on ancient peoples affected by disease-causing pathogens (Greenwood et al., 2001; Zink et al., 2002; Zink et al., 2005) such as Mycobacterium tuberculosis (Nerlich et al., 1997; Zink et al., 2001), Yersinia pestis
(Drancourt et al., 1998) and Plasmodium falciparum (Taylor et al.,
1997), or parasites such as nematodes (Ferreira et al., 1993), trypanosomes (Ferreira et al., 2000) and Ascaris (Loreille et al., 2001).
The study of ancient plant DNA has also showed several interesting outcomes, such as the hypothesis that ancient Ice Age climates have
10 influenced the extant genetic structure of plants (Lascoux et al., 2004;
see review by Gugerli et al., 2005).
Table 1.2. Historical or scientific relevance of recent findings within the field of molecular archaeology, where ancient DNA sources were used as the primary research material.
aDNA Source Historical/Scientific Relevance Reference Micrococcus luteus bacterial Investigated the ability of non- Greenblatt et al. species from 120-million-year- sporulating bacteria from the (2004) old amber Micrococcus genus to survive in extreme, nutrient-poor environments. Woolly mammoth dentine, rib, Investigated the evolution of Greenwood et al. tibia (4,500-26,000BP*) retroviruses prevalent in present (2001) mammals and humans Extinct bison (36,000BP*)† The first time a direct association Loy and Dixon Extinct moose (32,000BP*)† was made between the use of fluted (1998) Mammoth (21,000BP*)† projectile points and human Llano complex projectile points predation of extinct fauna and other (up to 9,500BP*) large Pleistocene mammals in arctic and sub-arctic North America. Teeth from 16th-18th century Confirmed the historic Black Death Drancourt et al. French skeletons of people plague by molecular identification (1998) thought to have died from the of Yersinia pestis. Plague *BP = years before present, obtained from calibrated 14C radiocarbon dates. † Samples consisted of preserved permafrost tissue.
The two major problems experienced in ancient DNA analysis include
the low amount of recoverable DNA and its degraded nature. Ancient
DNA can be recovered with some degree of difficulty from a variety of
samples, such as ancient bone (Zink et al., 2001; Ricaut et al., 2005),
teeth (Drancourt et al., 1998; Ricaut et al., 2005), fossilised materials
(Greenblatt et al., 2004), museum specimens (Higuchi et al., 1984),
tissue (Pääbo et al., 1988), and mummified remains (Pääbo, 1985;
Ferreira et al., 2000); as residues on ancient tools (Loy and Dixon,
1998), ancient implements or ancient household items such as pottery
11 jars (Cavalieri et al., 2003); parchments such as the Dead Sea Scrolls
(Woodward et al., 1996); pictographs or rock paintings (Reese et al.,
1996); and coprolites (Ferreira et al., 1993; Lorreile et al., 2001), amongst others. Ancient DNA is a very limiting resource and is considered precious due to the large amount of time and effort that is often required for its isolation. Thus, the type and number of tests performed on an ancient DNA sample is restricted by sample quantity and quality.
1.2.2.3. Limiting samples in the field of clinical diagnostics
The ability to analyse small sample quantities down to the single-cell level is desirable in the field of clinical diagnostics. Pathology testing of single-cell samples would be advantageous in the field of prenatal or preimplantation genetic diagnosis (PGD). The detection of DNA sequence copy number imbalances is usually performed via comparative genomic hybridisation (CGH), a process whereby fluorochrome-labelled test patient DNA is hybridised to human metaphase chromosomes against an equimolar amount of normal reference sample labelled with a different fluorochrome (Kallioniemi et al., 1992) (Figure 1.2).
CGH is a very powerful recent development of fluorescence in situ hybridisation (FISH). It is a molecular cytogenetic technique that is able to identify excess and missing chromosomal material that is often unresolvable by common G-banding techniques (Levy and Hirschhorn,
12 2002). The advantage of CGH over conventional FISH and whole chromosome painting (WCP) lies in its ability to identify not only the origin of extra or missing chromosomal material, but also maps the chromosomal position of the lost or gained material to specific chromosomal G-bands (Levy and Hirschhorn, 2002).
Figure 1.2. Simplified schematic of the comparative genomic hybridisation (CGH) protocol. Probes are generated by subjecting test/patient DNA to a nick-translation reaction, whereby the test DNA is labelled with a green fluorochrome and fragmented to between 300-1,000bp. Purified test probes are subsequently precipitated together with an equimolar amount of reference DNA probes labelled with red fluorochromes. The mixture of probes is then allowed to hybridised to immobilised normal human metaphase chromosomes on a glass microscope slide at 37°C for 2 days. Individual metaphase chromosomes are visualised using a fluorescence microscope with an attached CCD camera, with the aid of visualisation software on a computer. The software combines the total fluorescence from both test (green) and reference (red) DNA probes, displaying the result in a diagram featuring all 22 pairs of autosomal chromosomes plus the sex chromosomes. Excess green fluorescence at a particular region indicates an excess or gain of DNA in the test sample for that region, whereas an excess of red fluorescence indicates loss of chromosomal material in the DNA sample.
CGH allows the copy number of every chromosome in a cell to be determined in a single hybridisation and can detect changes present in as
13 little as 30-50% of the specimen cells (Kallioniemi et al., 1994), negating the need for aneuploidy screening from cultured cells. CGH is a unique technique that achieves whole-genome screening and is therefore significantly faster and less laborious than low-throughput methods for examining single dosage changes such as Southern analysis, PCR and
FISH (Beheshti et al., 2002). CGH can be performed either on a glass microscope slide containing human metaphases, or on a chip or microarray containing bacterial artificial chromosomes (BAC) or complementary DNA (cDNA) probes, which potentially increases resolution and throughput as particular chromosomes can be interrogated with higher discrimination. However, CGH does not reveal balanced translocations and inversions that do not change chromosome copy numbers (Levy and Hirschhorn, 2002).
Theoretically, CGH can be carried out on single cell samples. Routine applications of CGH, however, require approximately 0.2-1µg of genomic DNA (Wells and Levy, 2003). The ability to routinely analyse single cells from embryonic tissue resulting from natural conception or in vitro fertilisation for inherited diseases via CGH or other molecular techniques would revolutionise the field of clinical diagnostics.
Currently, the ability to reliably perform multiple tests on single cells is restricted due to the difficulty of amplifying limited DNA samples to a quantity and quality suitable for use in assays such as CGH. Samples can also be limiting in fields where large quantities of DNA are often available but are quickly depleted due to extensive high-throughput
14 analysis, such as in large-scale single nucleotide polymorphism (SNP) genotyping, genetic linkage analysis, genetic diversity studies, epidemiological investigations and also general laboratory research.
1.2.2.4. Overcoming the problem of limited samples
Overcoming the problems associated with the use of limited samples for
DNA testing requires a robust DNA amplification technology with several crucial properties to ensure reliable performance. Most importantly, the technology should be sensitive to low concentrations of
DNA template down to the single genome level, or even sub-genomic.
Secondly, the procedure should yield a large amount of DNA product that is compatible for use in downstream applications without the need for extensive manipulation or downstream processing. Thirdly, the product DNA should be of uniform quality to ensure that any results obtained from the analysis of the product DNA are representative of those from the template DNA, thus preventing the occurrence of misdiagnosis or conflicting results.
Many techniques have been devised to satisfy the above requirements and designed to amplify as much of the DNA sample as possible. The methods are primarily considered as a pre-amplification step, or an amplification method carried out on a limited sample prior to performing the DNA analysis of interest. These techniques are collectively known as whole genome amplification (WGA) methods.
15 1.3. Applying Whole Genome Amplification (WGA) Methods to
Overcome Problems with Limiting Sample Quantities
Whole genome amplification (WGA) methods are in vitro reactions devised to non-specifically amplify all of the genetic material present in samples containing low amounts of DNA, in order to provide sufficient
DNA template for molecular analysis. Ideally, any amplified DNA produced by a WGA technique would be representative of the original input DNA and perform identically to the DNA template material, generating test results that are indistinguishable. Much effort has been invested in developing methods for whole genome amplification in order to provide sufficient quantities of representative DNA to allow robust high-throughput molecular analyses.
Due to the non-specific nature of the process, WGA reactions will amplify all DNA sequences within the sample, including contaminating
DNA. Contaminating DNA is defined as DNA originating from sources other than the DNA sample of interest, and may include either human or non-human DNA sequences. It is thus a prerequisite to have appropriate measures to protect against contamination, such as the use of appropriate positive and negative controls within the WGA reaction and using assay- specific conditions in downstream applications.
WGA of human DNA is a challenging process, as it requires faithful amplification of more than 3 billion bases without the loss or preferential
16 amplification of any particular loci or alleles. According to Lasken and
Egholm (2003), important characteristics that should be possessed by a
WGA technique include (1) maximum yield or amplification fold; (2) the conservation of original template length in the final amplified product as this may affect applications such as restriction fragment length polymorphism (RFLP) analysis, cloning and sequencing; (3) fidelity and processivity of the DNA polymerase to ensure accurate replication of sample DNA for applications such as DNA sequencing and genotyping; (4) the ability to amplify small or trace DNA samples, including single cells; and (5) the scalability of the WGA reaction for preparative purposes.
Analysis of the available published literature suggests that there are predominantly two WGA approaches that are commonly used, namely
PCR-based protocols and non-PCR-based isothermal methods. Several
WGA methods employ protocols based on PCR using Taq DNA polymerase, such as degenerate oligonucleotide-primed PCR (DOP-
PCR) (Telenius et al., 1992) and primer extension preamplification
(PEP) (Zhang et al., 1992). PCR-based WGA methods differ from conventional PCR in that they aim to amplify all of the DNA sequences in a sample, in contrast to region-specific amplification as performed by
PCR. The two most common isothermal WGA methods include rolling circle amplification (RCA) by large fragment Bst DNA polymerase (Fire and Xu, 1995) or multiple displacement amplification (MDA) utilising highly processive bacteriophage φ29 DNA polymerase (also known
17 commercially as phi29 DNA polymerase) in conjunction with modified random hexamers (Dean et al., 2002).
1.3.1. PCR-based Whole Genome Amplification
The polymerase chain reaction (PCR) is a fast and convenient technique that amplifies a specific DNA region of interest as defined by the oligonucleotide primers used (Saiki et al., 1985; Mullis and Faloona,
1987). Typically, a DNA sample is denatured into single strands and incubated with DNA polymerase, deoxynucleotide triphosphates
(dNTP’s), and two oligonucleotide primers whose sequences flank a
DNA segment of interest. The DNA polymerase used is typically Taq
DNA polymerase, isolated from the thermophilic bacterium Thermus aquaticus. The PCR process involves multiple cycles of heating and cooling. In each cycle, heating separates double-stranded DNA, primers are annealed to complementary segments on the DNA, and DNA polymerase directs synthesis of the complementary DNA sequence between the two primers. Multiple cycles of this process is performed, with each cycle doubling the amount of target DNA from starting material as little as a single cell.
It must be noted that PCR by Taq DNA polymerase is not an error-free process due to the Taq error rate of approximately 3x10-4 (Eckert and
Kunkel, 1991), the introduction of AT-to-GC transitions (Keohavong and Thilly, 1989) as well as deletion mutations due to DNA secondary
18 structures (Cariello et al., 1991). The PCR reaction was found to accumulate about one mutation per 900 bases after 20 cycles that can be compounded in subsequent cycles (Saiki et al., 1988). However, there are now a multitude of other DNA polymerases with lower error rates that can be used for PCR applications, such as those originating from
Pyrococcus sp. (Uemori et al., 1993; Uemori et al., 1997; Takagi et al.,
1997; Dietrich et al., 2002) or mixtures of Taq and an additional proofreading DNA polymerase such as Pwo or Tgo DNA polymerases, as available in commercial kits such as the Expand High Fidelity PCR
System (Roche Diagnostics, Mannheim, Germany). A detailed discussion on Taq DNA polymerase is outside the scope of this thesis, and readers are directed to the above references.
DNA amplification techniques that are modifications of the Taq DNA polymerase PCR process, in order of their publication dates, include
Alu-PCR (Nelson et al., 1989), linker adapter PCR (Ludecke et al.,
1989), degenerate olignonucleotide-primed PCR (Telenius et al., 1992), primer extension preamplification (Zhang et al., 1992) and tagged random primer PCR (Grothues et al., 1993). In the following discussion, more focus is given to DOP-PCR and PEP, as references to their applications in WGA are more extant in the literature.
19 1.3.1.1. Alu-PCR, Linker Adapter PCR (LA-PCR) and Tagged Random
PCR (T-PCR)
Alu-PCR and linker adapter PCR are concerned with the isolation of regions of DNA from complex DNA/protein sources or the cloning of microdissected chromosomal regions respectively, and are not widely utilised for whole genome amplification purposes per se. Alu-PCR, also known as interspersed repetitive sequence (IRS) PCR, involves region- specific amplification of repetitive Alu repeats that are interspersed throughout the human genome (Nelson et al., 1989). Alu-PCR relies on the assumption that Alu repeats are distributed evenly across the human genome. However, Alu repeats are clustered in G (light) chromosome bands (Korenberg and Rykowski, 1988) and therefore these regions are predisposed to amplification bias.
In contrast to Alu-PCR, linker adapter PCR (LA-PCR), also known as ligation-mediated PCR (LMP), requires the digestion of DNA in microdissected chromosomes by a frequent blunt-cutting restriction endonuclease, usually Mse I, followed by ligation of a non-palindromic double-stranded linker with an overhanging end to the blunt ends, thus priming the amplification reaction using one primer species (Ludecke et al., 1989). Mse I restriction sites are randomly present in about every
200-300bp of human DNA (Klein et al., 1999), which makes LA-PCR suitable for the amplification of degraded DNA samples (Pirker et al.,
2004). By using a single primer species for subsequent PCR
20 amplification, LA-PCR avoids the problem of sequence complexities for multiple binding sites that is encountered in multi-primer techniques such as DOP-PCR and PEP. Unlike Alu-PCR, LA-PCR does not introduce significant amplification bias because of clustered primer binding sites, but the length of product generated is shortened due to restriction digestion of template DNA and thus only a limited subset of sequence is amplified. However, LA-PCR involves more steps than a conventional PCR reaction and multiple tube transfers, therefore more opportunities exist for the failure or loss of sample.
Since the introduction of the Ludecke et al. LA-PCR method (Ludecke et al., 1989), several variations of the protocol have been developed. An adapter/ligation-based WGA method termed single cell comparative genomic hybridisation (SCOMP) (Klein et al., 1999) has been shown to successfully amplify single-cell samples for use in comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH).
Instead of microdissected chromosome bands, SCOMP utilises genomic
DNA digested with Mse I, creating a high complexity mixture of fragments less than 2kb in size (Klein et al., 1999). SCOMP includes a pre-annealing step, whereby two oligonucleotides form an adapter complex that is then ligated to the overhangs on digested DNA fragments. Like LA-PCR, a single primer species is used to amplify all of the DNA templates in the sample. To avoid template loss, all preparatory steps were performed in a single tube (Klein et al., 1999).
21 Another adaptation of LA-PCR technology involves ligating primer sequences onto randomly-sheared genomic DNA in a process termed adapter-ligation PCR of randomly sheared genomic DNA (PRSG)
(Tanabe et al., 2003), to generate template DNA from single human cells for use in microsatellite genotyping and array-CGH. The difference between SCOMP and PRSG primarily lies in the method of fragmenting the DNA sample prior to ligation of adapters. Instead of enzymatic digestion of genomic DNA, PRSG utilises an automated instrument to perform hydrodynamic shearing of the DNA. The hydrodynamic procedure for shearing DNA is a time-consuming step, however the authors claim that the mechanical method creates fragments that minimise biased amplification resulting from differences in template sequences, such as high GC contents that can form stable secondary structures, which often prevents PCR amplification at standard annealing temperatures (Tanabe et al., 2003).
To a certain extent, tagged-random PCR is applicable to whole genome amplification. Tagged-random PCR (T-PCR) involves a two-step protocol using random primers with degenerate 3’ ends and a constant 5’ tail (Grothues et al., 1993). The first stage of the non-specific amplification reaction employs the degenerate primers, followed by removal of unbound primers and a second amplification stage using primers specific to the 5’ tail (Grothues et al., 1993). Although the system was sensitive down to picogram amounts of DNA and did not produce non-specific artefacts (Grothues et al., 1993), the two-step
22 strategy in T-PCR may jeopardise throughput and also introduces problems such as loss of DNA and the occurrence of contamination.
1.3.1.2. Degenerate oligonucleotide-primed PCR (DOP-PCR)
The degenerate oligonucleotide-primed PCR (DOP-PCR) method employs a degenerate primer (5’-CCGACTCGAGNNNNNNATGTGG-
3’) 22bp in length that binds to approximately one million sites in the human genome during several low-temperature cycles in PCR (Telenius et al., 1992). Only the fragments that are tagged with the specific sequences of the primer are amplified in a subsequent extension cycle at a higher temperature. The PCR cycle is repeated several times to achieve exponential amplification of tagged sites. The resulting PCR product is a mix of amplicons of varying lengths covering the whole genome. DOP-
PCR products may be used as template for subsequent DOP-PCR reactions, therefore providing a limitless amount of DNA.
DOP-PCR is less technically challenging than LA-PCR and the use of degenerate primers overcomes the limitations of Alu-PCR. DOP-PCR is a well established and widely accepted WGA method that has found applications in many genetic assays, including the investigation of chromosomal imbalances from tumour specimens by comparative genomic hybridisation or CGH (Kuukasjärvi et al., 1997; Kim et al.,
1999; Huang et al., 2000; Larsen et al., 2001; Pirker et al., 2004), SNP genotyping (Barbaux et al., 2001; Jordan et al., 2002; Grant et al.,
23 2002), microsatellite genotyping (Cheung and Nelson, 1996; Buchanan et al., 2000; Kittler et al., 2002), multi-locus detection of disease-causing mutations (Cheng et al., 1998) and the generation of DNA archives from precious anthropological samples (Buchanan et al., 2000). Especially significant was the use of DOP-PCR for the genotyping of low- concentration DNA samples for clinical diagnostics and SNP analysis
(Kuukasjärvi et al., 1997; Barbaux et al., 2001). DOP-PCR was also used for the reduction of genome complexity for SNP genotyping analyses of a variety of complex eukaryotic genomes such as human, mouse and Arabidopsis thaliana, effectively allowing the completion of a genome-wide SNP scan with minimal product required, thus opening the way for high-throughput genotyping (Jordan et al., 2002).
Furthermore, the DOP-PCR WGA method has been coupled with the use of silicon-glass chips for the locus-specific, multiplex PCR of dystrophin gene exons in the detection of deletions causing
Duchenne/Becker muscular dystrophy (Cheng et al., 1998).
Fluorescence in situ hybridisation (FISH) analysis of a biotinylated
DOP-PCR product showed satisfactory genome coverage (Telenius et al., 1992). At the time, DOP-PCR became the method of choice for amplifying minute DNA samples for the detection of DNA sequence copy number imbalances in comparative genomic hybridisation (CGH).
When high quality samples were amplified, DOP-PCR generated DNA suitable for use in CGH. The applicability and quality of DOP-PCR products have also been assessed in the amplification of whole genomic
24 DNA for CGH analysis of partly degraded or limited samples derived from different sources such as melanoma and hepatocellular carcinoma cell cultures, frozen tissues and paraffin-embedded tissues but was prone to false-positive and/or false-negative results (Pirker et al., 2004). The success rate of amplifying DNA from formalin-fixed, paraffin embedded samples by DOP-PCR is dependent on immediate handling of the tissue post-surgery and length of fixation time (Yagi et al., 1996; Dietmaier et al., 1999).
DOP-PCR is capable of amplifying template DNA from concentrations as low as 25pg or the equivalent of four normal diploid human cells
(Kuukasjärvi et al., 1997). The amplification of DNA samples less than
20pg was shown to cause a series of false-positive and false-negative results in CGH, characterised by a decrease in gained regions and increase of lost regions (Pirker et al., 2004). Voullaire et al. (1999) demonstrated that DOP-PCR-amplified product from a normal control sample displayed under-representation and false-deletions in heterochromatic regions during CGH. Similar observations were also made in other studies (Huang et al., 2000; Larsen et al., 2001).
Increasing the number of PCR cycles in an optimised DOP-PCR protocol was shown to increase sensitivity of the reaction, enabling the amplification of small amounts of DNA as low as 12.5pg without affecting yield and amplification bias for use in CGH (Huang et al.,
2001). However, it is generally accepted that starting a DOP-PCR
25 reaction with less than 1ng of gDNA template severely reduces fidelity and increases the chance of allelic dropout (Kittler et al., 2002).
The yield of a DOP-PCR reaction is consistent for samples of similar concentrations but is dependent on the total concentration of degenerate primers used (Barbaux et al., 2001). For starting materials of 10, 30 or
50ng of DNA in a 50µL DOP-PCR reaction, the final yield was increased from 5µg to 7µg when primer concentration was increased from 100pmol to 200pmol (Barbaux et al., 2001). Earlier studies have shown that DOP-PCR can produce up to 23,000 times as much of the starting amount of DNA, but it was acknowledged that the majority of
DNA generated was due to non-specific amplification and never amplified as well as the original sample (Cheung and Nelson, 1996). A more acceptable limit for the yield of a DOP-PCR reaction is approximately 500-fold from 10ng of template DNA, sufficient for approximately 400-500 PCR assays (Bannai et al., 2004; Barbaux et al.,
2001; Cheung and Nelson, 1996).
DOP-PCR products appear as smears on agarose gels stained with ethidium bromide, containing fragments ranging from between 200 and
2000bp in length, with an average size of approximately 300-600bp
(Larsen et al., 2001; Barbaux et al., 2001). The small amplimers produced by DOP-PCR limit their use in applications that require the conservation of original template length as necessitated by RFLP analysis and DNA sequencing. Optimisation of the DOP-PCR protocol
26 and substitution of the Taq DNA polymerase by a modified, thermostable bacteriophage T7 DNA polymerase known as
ThermoSequenase increased the length of WGA products to within a range of 300-4,000bp (Kuukasjärvi et al., 1997; Huang et al., 2000).
Product sizes were increased further to about 7,000bp-10,000bp with the use of a high-fidelity long-range PCR polymerase mix of Taq DNA polymerase with proofreading Pwo DNA polymerase in a reaction termed long-DOP-PCR or LL-DOP-PCR (Buchanan et al., 2000; Kittler et al., 2002), although this modification did not improve the utility of amplified degraded DNA templates in short tandem repeat (STR) typing
(Kittler et al., 2002).
A recent study has identified the presence of up to 1,000,000-fold amplification bias in DOP-PCR products compared to the original starting DNA as determined by TaqMan quantitative PCR (Dean et al.,
2002). The presence of such a high degree of bias has significant implications for the use of DOP-PCR products in assays requiring representative WGA templates, especially for use in diagnostic assays such as CGH. The use of DOP-PCR products in SNP genotyping resulted in less stable data clusters and an increase in ambiguous genotype calls of 5.8% compared to 2% for the original gDNA (Grant et al., 2002). DOP-PCR SNP assays also displayed a higher overall error rate of 0.7% compared to the original gDNA, manifested as allelic loss or loss of heterozygosity that could not be attributed to differences in quality of the original DNA samples (Grant et al., 2002).
27
Allelic drop-ins, or the creation of additional alleles, were also observed in DOP-PCR and were attributed to cross-contamination which accrued during subsequent PCR cycles (Grant et al., 2002). Due to the low fidelity of Taq DNA polymerase used in DOP-PCR, errors can be accumulated during the WGA reaction and thus introduces the possibility of misdiagnosis in limited DNA samples such as single cells.
1.3.1.3. Primer Extension Preamplification (PEP)
Primer extension preamplification (PEP) was first utilised to amplify
DNA from single sperm nuclei to generate sufficient DNA template for analysis (Zhang et al., 1992). The PEP protocol involves the use of totally degenerate 15bp oligonucleotides (5’-NNNNNNNNNNNNNNN-
3’) and Taq DNA polymerase in 50 PCR cycles with each cycle consisting of a 1 minute denaturation step (92°C), low-stringency primer annealing (37°C), a gradual 10s/°C ramp up to 55°C followed by a 4 minute elongation step (55°C). Zhang et al. (1992) estimated that at least
78% of genomic DNA in a single human haploid cell could be copied no less than 30 times by PEP, with an average of 60 copies per PEP reaction. It was postulated that modifications to the PEP protocol affecting primer length, primer extension, dNTP concentration or other constituents might increase the yield of the PEP reaction (Zhang et al.,
1992).
28 An optimised PEP protocol was introduced and termed improved-PEP or
I-PEP (Dietmaier et al., 1999). I-PEP differs from the original PEP protocol in the use of a higher concentration of Taq DNA polymerase with an additional Pwo proofreading DNA polymerase as used in LL-
DOP-PCR, and increasing the elongation temperature from 55°C to
68°C (Dietmaier et al., 1999). I-PEP was found to be superior to the original PEP protocol and the established DOP-PCR method for amplification of DNA from a single cell, with I-PEP success rate of 40% compared to 15% for PEP and 3% for DOP-PCR (Dietmaier et al.,
1999). Success rates for the amplification of DNA from five cells were typically 100%, 33% and 20% for I-PEP, PEP and DOP-PCR respectively (Dietmaier et al., 1999). For formalin-fixed, paraffin- embedded tissues, I-PEP was also shown to be superior to PEP, yielding generous products from 30-cell clusters whereas the original protocol failed to amplify samples containing up to 1,000 cells (Dietmaier et al.,
1999).
PEP has been utilised in many applications as an alternative to DOP-
PCR. PEP was reported to be more reliable than DOP-PCR for certain applications. Specifically, the I-PEP method has been utilised for microsatellite instability and loss of heterozygosity analysis in 1 to 1,000 cells (Dietmaier et al., 1999) and successful mutation detection in single cell samples from ethanol-fixed, paraffin-embedded tissues (Dietmaier et al., 1999; Heinmöller et al., 2002). I-PEP was also applied to high- throughput allelotyping of limited archival tumour tissue samples for
29 loss of heterozygosity analysis (Wang et al., 2001). PEP of buccal swabs was performed for use in downstream SNP assays but was found to generate a large number of failed PCR’s (Anchordoquy et al., 2003).
However, applying I-PEP for WGA of buccal cell samples improved subsequent PCR, decreasing failure rates from 30-57% to 2-5%, before and after I-PEP respectively (Zheng et al., 2001).
Small product sizes, allelic drop-out, and low yields still remain as obstacles that inhibit the wide use of PEP in clinical diagnostics.
Amplification of ~500bp segments occurred with a success rate of 68%, whilst 2kb fragments could be amplified 8% of the time when good quality, high molecular weight DNA was used as template (Heinmöller et al., 2002). The generation of longer products by PEP or I-PEP has not been demonstrated. However, it is hypothesised that any PEP-based method is not able to generate long amplification products, as the completely degenerate primers can anneal to internal regions within fragments amplified in previous cycles, therefore generating shorter amplicons as the reaction proceeds (Kittler et al., 2002). I-PEP of one buccal swab may yield sufficient template for up to 900 PCR reactions
(Zheng et al., 2001).
The use of Pwo polymerase with proofreading activity in combination with Taq polymerase minimised artificial deletions and transitions during the I-PEP reaction, and 100% sequencing accuracy was established for greater than 4,000 sequenced base pairs (Dietmaier et al.,
30 1999). However, PCR amplicons in this study were less than 200bp in length, and sequence fidelity in fragments greater than 600bp was not assessed. The allelic drop-out rate for I-PEP of single cells was 68%, but was not observed when five or more cells were used (Heinmöller et al.,
2002). Preferential amplification was also encountered in single cell I-
PEP reactions, although this effect was reduced when a minimum of 10 cells were introduced into the reaction (Dietmaier et al., 1999).
Amplification bias in PEP products was recently established to be within the 104-fold range (Dean et al., 2002), and may therefore not be appropriate for use in disease diagnostics or allelic imbalance analysis of clinical samples, especially single cell or fixed tissue samples, or forensic casework samples. Furthermore, the low yields of PEP reactions from single cells restrict high-throughput applications and the archiving of limited samples. WGA of 5ng gDNA by I-PEP typically produces sufficient DNA template for 100 PCR reactions, compared to 500 PCR assays generated by DOP-PCR of 10ng samples (Bannai et al., 2004).
1.3.1.4. Summary on the characteristics of PCR-based WGA methods
PCR-based WGA methods attempt to overcome the problem of limiting sample, but generate low amplification yields of typically less than
1,000-fold (Bannai et al., 2004; Barbaux et al., 2001; Cheung and
Nelson, 1996; Zheng et al., 2001). These amounts may be suitable for confirmatory testing but would not suffice for archival storage or for use
31 in large scale high-throughput genotyping studies. Furthermore, PCR- based WGA products are typically less than 600bp in length (Larsen et al., 2001; Barbaux et al., 2001; Heinmöller et al., 2002), and in the case of PEP, shorter fragments are generated in subsequent cycles as primers anneal within regions internal to amplified products from previous cycles (Kittler et al., 2002). Modifications to original protocols have allowed the amplification of products up to 10kb by LL-DOP-PCR, and issues pertaining to the fidelity of Taq DNA polymerase were overcome by introducing the use of proofreading enzymes such as Pwo DNA polymerase in combination with the Taq.
Preferential amplification or allelic dropout of certain loci can occur during PCR due to factors such as template length, secondary structure and GC content (Lasken and Egholm, 2003). This results in significant bias of up to 1,000,000-fold between different loci within the same DNA sample (Dean et al., 2002). The identification of false-positives and false-negatives are frequent in PCR-based WGA products, especially in the amplification of low copy number DNA samples such as single cells or partly degraded samples (Pirker et al., 2004; Voullaire et al., 1999;
Huang et al., 2000; Larsen et al., 2001). The accrual of artificial deletions (Keohavong and Thilly, 1989; Cariello et al., 1991) by low- fidelity Taq DNA polymerase can promote generation of non- representative DNA and amplification errors that can cause subsequent distortion in testing results, leading to misdiagnosis of patient samples or
32 misidentification of individuals in crime scene samples analysed by PCR of STR loci.
Despite much effort in the optimisation of PCR-based WGA, it must be acknowledged that the use of short products with questionable allelic balance can be problematic in applications that depend on longer representative WGA products such as genome-wide SNP studies, whole genome allelic imbalance assays such as CGH, and STR profiling or identification of forensic specimens. WGA methods that overcome many of the problems associated with PCR-based WGA have been described recently and are now receiving increased attention from the research and diagnostic industries because of the many advantageous characteristics that are discussed in the following sections.
1.3.2. Non-PCR-based isothermal Whole Genome Amplification by strand-displacing Bst and φ29 DNA polymerases
An alternative approach to WGA uses isothermal strand-displacing DNA polymerases in a mechanism that is not based on the PCR process.
Strand-displacement activity is an inbuilt mechanism that prevents the introduction of errors and duplications due to polymerase slippage and misalignment of two DNA strands during replication (Canceill et al.,
1999). During amplification of linear templates with the use of random primers, strand-displacement allows the displacement of the 5’ end by another upstream strand polymerising in the same direction during DNA
33 synthesis, thereby liberating single-strand DNA product for further priming and extension and therefore generating high molecular weight hyperbranched structures (Lage et al., 2003). DNA polymerases that possess some degree of strand-displacing activity include phage M2,
Vent™, Klenow fragment, Sequenase™, T5, PRD1, T4, Bst and φ29
DNA polymerases (Lizardi U.S. patent 6,124,120, 2000).
The more common isothermal WGA reactions are performed by either using a cocktail containing large fragment Bst DNA polymerase and a single-strand DNA binding protein, or by utilising bacteriophage φ29
DNA polymerase. Although both systems depend on strand- displacement activity, differences in terms of polymerase fidelity and yield exist. The isothermal WGA protocol involves a short DNA denaturation step at 94-95°C, followed by incubation at a constant temperature that is specific for the DNA polymerase for 6-18 hours, with a final high temperature incubation for 10-15 minutes to inactivate the enzymes (Dean et al., 2002; Lage et al., 2003).
1.3.2.1. Rolling Circle Amplification (RCA) by Large Fragment Bst DNA polymerase
Rolling circle amplification (RCA) is a natural process intrinsic to some microorganisms for the replication of circular DNA molecules such as plasmids or viral genomes (Kornberg and Baker, 1992). This process was adapted for in vitro amplification of circular DNA templates using
34 Bst DNA polymerase I. Bst DNA polymerase I originates from the moderately thermophilic bacteria Bacillus stearothermophilus, which grows optimally at 65-70°C, and was first reported in 1972 (Stenesh and
Roe, 1972). Early exonuclease assays demonstrated that Bst DNA polymerase I possessed dsDNA-dependent 5’→3’ exonuclease activity for degrading Okazaki fragments during DNA synthesis and excising lesions during DNA repair, but lacked 3’→5’ exonuclease activity for proofreading and removal of mismatched nucleotides (Stenesh and Roe,
1972; Aliotta et al., 1996). The loss of proofreading activity was theorised to be an evolutionary consequence of enhancing thermostability in thermophilic organisms (Aliotta et al., 1996).
The N-terminal domain, containing 5’→3’ exonuclease activity, can be separated from the polymerisation domain to form a 75kDa DNA polymerase domain termed the ‘large fragment’, which is comparable in function and characteristics to the Klenow fragment of E. coli polymerase I (Ye and Hong, 1987; Aliotta et al., 1996). The 3’→5’ exonuclease (-) Klenow fragment has a reported error rate of approximately 1x10-4 (Bebenek et al., 1990), similar to the error rate for
Taq DNA polymerase as used in PCR (Eckert and Kunkel, 1991).
In vitro RCA involves annealing a single primer to circular template
DNA and in conjunction with large fragment Bst DNA polymerase promotes continuous elongation of the circle, synthesising tandem copies of the template (Fire and Xu, 1995; Lizardi et al., 1998). Hyperbranched
35 RCA is accomplished by using two primers for each DNA strand, generating exponential amplification of the circular DNA by a cascade of strand displacement reactions (Lizardi et al., 1998). The use of random hexamers also allows synthesis of both strands, to produce double-stranded product during cascading hyperbranched amplification
(Dean et al., 2001).
Large fragment Bst DNA polymerase is the enzyme of choice for efficient RCA due to inherent SDA activity. This DNA polymerase performs WGA at a constant temperature between 50-65°C (Aliotta et al., 1996; Viguera et al., 2001; Lage et al., 2003). DNA quantitation assays indicate that isothermal WGA by large fragment Bst DNA polymerase after a 5-hour incubation can amplify template DNA by approximately 250-fold, and prolonged incubations greater than 5 hours result in greater than 1,000-fold amplification to generate high molecular weight products greater than 10,000bp (Lage et al., 2003).
An evaluation of amplification bias in Bst WGA products by human cDNA array-CGH did not reveal any significant over- or under- representation of chromosomal regions in amplified human gDNA samples, with ratios between unamplified and amplified loci close to 1:1
(Lage et al., 2003). Quantitative TaqMan PCR assays also found no significant differences in sequence representation between human chromosomal DNA and circular mitochondrial DNA (Lage et al., 2003).
36 The lack of bias in Bst WGA products contrasts that displayed by PCR- based WGA products as discussed earlier.
Isothermal WGA by large fragment Bst DNA polymerase is an efficient method for the amplification of limited DNA samples for further analysis and storage. Bst WGA yields sufficient amounts of high molecular weight DNA with no apparent bias. However, the lack of
3’→5’ exonuclease activity for proofreading during DNA polymerisation contributes to the relatively high error rate for Bst polymerase. The absence of proofreading activity is a significant disadvantage for RCA by Bst DNA polymerase, particularly for use in applications such as forensic human identification or disease diagnostics.
1.3.2.2. Multiple Displacement Amplification (MDA) by φ29 DNA polymerase
Multiple displacement amplification (MDA) is a recently introduced isothermal WGA technique utilising the properties of φ29 DNA polymerase for representative amplification of circular and linear genomic templates (Dean et al., 2002). MDA is based on rolling circle amplification technology coupled with the very strong strand displacement activity of the φ29 DNA polymerase.
The strand-displacing φ29 DNA polymerase is a 68kDa monomeric enzyme encoded by the lytic Bacillus subtilis bacteriophage φ29 for
37 replication of its double-stranded linear genome (Watabe et al., 1984).
Early studies have shown that φ29 DNA replication occurs via a protein- primed mechanism whereby DNA synthesis proceeds non- simultaneously from either end of the linear DNA molecule, and subsequent elongation of the DNA chain occurs via a strand displacement mechanism as described above (Watabe et al., 1984;
Blanco and Salas, 1985; Blanco et al., 1989). The protein-priming mechanism involves the covalent linkage of the 30kDa φ29 terminal protein p3 to each 5’-end by a phosphodiester bond between the OH- group of serine 232 and 5’-dAMP at both ends of the φ29 genome
(Geiduschek and Ito, 1982; Zaballos and Salas, 1989; Blanco and Salas
1996; Rodríguez et al., 2004; Truniger et al., 2004). It was later discovered that φ29 DNA polymerase was able to perform synthesis of foreign DNA in vitro without the assistance of protein-priming, using instead a single oligonucleotide primer annealed to M13 DNA (Blanco et al., 1989). This technique has been adapted for the amplification of circular and linear DNA templates using exonuclease-resistant, thiophosphate-modified random hexamers (5’-NpNpNpNpNpNp-3’) in the isothermal WGA method termed multiple displacement amplification or MDA (Dean et al., 2001; Dean et al., 2002) (Figure 1.3).
38
Figure 1.3. A schematic of the multiple displacement amplification (MDA) reaction. During incubation at 30°C, random hexamers bind to denatured, single-stranded template DNA and are extended by φ29 DNA polymerase. The 5’-end of each growing strand is displaced by another upstream strand, liberating single-strand DNA available for new random priming events. The branched network expands dramatically as the reaction proceeds (adapted from Dean et al., 2002).
MDA by φ29 DNA polymerase involves incubating φ29 DNA polymerase, dNTP’s, random hexamers and denatured template DNA at
30°C for 16-18 hours. The enzyme is inactivated at 65°C for 10 minutes, and the product DNA can be used directly in downstream applications.
The φ29 DNA polymerase carries out highly processive and continuous elongation of each DNA strand, and coupled with strong strand displacement activity, results in the synthesis of long fragments in isothermic conditions (Blanco et al., 1989; Esteban et al., 1993; Murthy et al., 1998; de Vega et al., 1999; Zhang et al., 2001). It must be noted that φ29 DNA polymerase possesses greater strand-displacing activity than the Bst DNA polymerase cocktail (Lage et al., 2003). At optimum conditions, a single φ29 DNA polymerase initiates replication and
39 elongation of DNA without dissociating from the template at the rate of approximately 40 nucleotides/second, with multiple copies of the full- length template exceeding 70kb after a short 40-minute incubation
(Blanco et al., 1989). Elongation of the same DNA template by Klenow fragment was not able to effectively conserve the full-length template, as the enzyme stopped and dissociated at specific multiple sites during
DNA synthesis (Blanco et al., 1989). Increasing dNTP concentration in an in vitro assay established a higher φ29 DNA polymerase reaction rate of approximately 53 nucleotides/second, with the generation of products greater than 23kb in length within a 10-minute incubation period
(Lizardi et al., 1998). Sequence analysis of cloned amplification products revealed conservation of all sequences from the original template DNA (Lizardi et al., 1998).
DNA polymerisation catalysed by φ29 DNA polymerase is a highly accurate process. The φ29 DNA polymerase fidelity mechanism couples nucleotide insertion discrimination, or the preferential selection of the correct dNTP for phosphodiester bond formation, with strong 3’→5’ exonucleolytic proofreading activity to result in a very low error rate of approximately 1x10-6 to 1x10-7 (Esteban et al., 1993). The 3’→5’ exonuclease active site is located in the N-terminal domain of the DNA polymerase, with polymerisation activity localised in the C-terminal domain (see review of φ29 DNA polymerase structure and function by
Blanco and Salas, 1996). Studies have identified that in a fully-folded and actively-synthesising enzyme, the distance between the enzyme
40 surface boundary to the exonuclease and polymerisation sites are 5 and 6 nucleotides respectively, suggesting a cleft the size of only one nucleotide separating polymerisation and proofreading activities (de
Vega et al., 1999). This spatial arrangement of structure and function allows φ29 DNA polymerase to easily switch between polymerisation and editing modes during DNA synthesis using a sliding-back mechanism, without dissociating from the template DNA (de Vega et al.,
1999).
As a result of its processivity and intrinsic strand-displacing activity,
MDA using φ29 DNA polymerase is able to generate microgram quantities of DNA from sub-picogram amounts of starting template. The yield of amplified DNA is consistent regardless of starting material between 100fg and 10ng, producing 20-30µg of high molecular weight product in a 100µl scalable reaction within 6 hours (Dean et al., 2002).
MDA is able to amplify DNA samples by up to 100,000-fold from 0.3ng of DNA, the equivalent of 46 human diploid cells (Dean et al., 2002).
MDA performed directly on cell lysates from clinical samples such as buccal cells, whole blood, finger-stick blood and Guthrie cards produced reproducible yields of about 10,000-fold amplification regardless of the original amount of sample amplified (Hosono et al., 2003). MDA was also performed on approximately 10 tissue culture cells to generate similar yields (Dean et al., 2002). These amplification folds are much greater than those encountered in PCR-based WGA methods as described earlier.
41
Products generated by MDA were analysed to determine sequence fidelity and representative coverage of the original template DNA using a variety of methods such as STR typing (Hosono et al., 2003), SNP genotyping (Hosono et al., 2003; Tranah et al., 2003), SNP chip analysis
(Paez et al., 2004; Lovmar et al., 2003; Barker et al., 2004), direct sequencing (Paez et al., 2004), comparative genomic hybridisation
(CGH) (Dean et al., 2002), cDNA microarrays or array-CGH (Lage et al., 2003; Wang et al., 2004), and quantitative TaqMan assays (Dean et al., 2002; Hosono et al., 2003; Wang et al., 2004). Results from the analyses are described below.
STR typing of 20 MDA products using the AmpFlSTR® Profiler Plus®
STR system, which is commonly used in forensic biology laboratories, produced allele calls that were 100% concordant with the original gDNA template (Hosono et al., 2003). The ability to perform SNP genotyping from MDA products is evidence of sequence conservation and representative amplification by this WGA method. Small-scale genotyping of 5 SNP loci on 20 MDA-amplified products from whole blood, finger-stick blood and buccal swabs also revealed 100% concordance with the original gDNA (Hosono et al., 2003). Genotyping of 6 SNP’s across four genes for a larger subset of 352 MDA-amplified samples demonstrated 99.95% accuracy (Tranah et al., 2003).
Minisequencing and microarray SNP analysis revealed 99.7% concordance of MDA products to gDNA, whilst PEP only scored 88.7%
42 concordance (Lovmar et al., 2003). Large-scale SNP genotyping across a 2,320-SNP linkage panel observed greater than 99.8% concordance
(Barker et al., 2004), a value that is identical to studies utilising high density Affymetrix 10K SNP oligonucleotide arrays probing more than
10,000 SNPs (Paez et al., 2004). Furthermore, high molecular weight
MDA products enabled successful RFLP genotyping of 16 random individuals for the presence of a 2.1kb and 1.1kb PstI fragment (Dean et al., 2002). MDA of 10 genomic copies also demonstrated successful restriction fragment analysis of a 1.9kb fragment within the human parathyroid gene (Dean et al., 2002).
CGH analysis of MDA products was used to determine uniformity of chromosomal coverage. Signal along the length of chromosomal arms indicated uniform coverage, but MDA-amplified products displayed reduced centromeric signals to indicate some degree of loss in repetitive centromeric sequences (Dean et al., 2002). The loss of some sequences was supported in another study by a combination of SNP genotyping and direct sequencing of MDA products, concluding that MDA is estimated to cover approximately 99.82% of the input gDNA, effectively providing comprehensive genome representation (Paez et al., 2004). However, this study did not attribute sequence loss to particular chromosomal regions or specific repetitive elements, but noticed amplification difficulties resulting in low intensity and poor SNP calls in six regions, namely
1q42, 4q35, 6p25, 7q36, 10q26 and 18p11 (Paez et al., 2004).
43 Microarray, high-resolution array-CGH and quantitative TaqMan PCR analyses have shown some degree of sequence misrepresentation in
MDA-amplified samples. MDA products have been reported to display up to 3-fold amplification bias, with an average representation of 80-
225% (Dean et al., 2002). This bias introduces a significant level of under- and over-representation of sequences compared to RCA by the large fragment Bst DNA polymerase and T4 gene 32 protein cocktail, which did not display any bias. TaqMan analysis of 47 loci in 44 patients identified amplification bias ranging between 0.5 to 3-fold (Hosono et al., 2003). It appeared that 3-fold bias remained relatively constant between 100- and 100,000-fold amplification (Dean et al., 2002).
Evaluation of MDA products using microarrays containing 4,592 human cDNA clones revealed deviation from the expected 1:1 ratio at numerous loci when compared to unamplified gDNA, in contrast to ratios obtained with Bst DNA polymerase that were close to 1:1 (Lage et al., 2003).
However, array-CGH genomic copy number screening of breast cancer cells amplified by MDA did not detect substantial bias and produced results comparable with unamplified DNA (Wang et al., 2004). This result appears to disagree with other published reports.
The inconsistency in MDA genome coverage data between STR typing and SNP genotyping versus whole genome microarray hybridisation is probably due to the fact that STR and SNP methods are quite narrow in their focus as they only examine several loci that are spatially distant.
This is in contrast to multilocus quantitative TaqMan assays or high-
44 resolution array CGH methods that provide a more holistic view of the genome, thus offering a better picture of amplification bias in DNA templates amplified by MDA.
MDA WGA has been utilised in a variety of clinical diagnostic applications, but utility in forensic biology is still limited. Since its conception, MDA-amplified materials have found utility in conventional
SNP genotyping (Hosono et al., 2003; Tranah et al., 2003) and SNP genotyping by minisequencing (Lovmar et al., 2003), real-time SNP detection on the BD ProbeTec system (Wang et al., 2003), using automated fluorescence correlation spectroscopy (Bannai et al., 2004), multiplex SNP analysis on the BeadArray genotyping platform (Pask et al., 2004), and using SNP chips or microarrays (Lovmar et al., 2003;
Paez et al., 2004; Barker et al., 2004; Huang et al., 2004). MDA also finds utility in clinical applications such as array-CGH for copy number imbalance analysis (Lage et al., 2003; Wang et al., 2004), preimplantation genetic diagnosis of single cells (Hellani et al., 2004), and the generation of unlimited amounts of DNA template from clinical specimens (Luthra and Medeiros, 2004). MDA is also gaining acceptance for other uses such as in molecular epidemiology (Yan et al.,
2004), the amplification of insect genomes from crude extracts
(Gorrochotegui-Escalante and Black, 2003), detection of bacteria from single eggs of Nesbitt mites (Jeyaprakash and Hoy, 2004) and amplification of DNA in residual cells deposited by incidental contact
(Sorensen et al., 2004).
45
The utility of MDA has not been fully assessed for use in applications such as sample archiving, forensics and single cell clinical diagnostics.
More importantly, methods for overcoming amplification bias in MDA- amplified products have not been described. This therefore limits the effective use of MDA templates in the analysis of pathology samples for clinical diagnostics, or in forensic investigations for the purposes of amplifying evidentiary material suitable for analysis and reporting in a court of law.
1.4. Research Rationale: Background to the Investigation
Many papers report the use of WGA for a variety of applications such as in genetic disease diagnosis, tumour/cancer analysis, prenatal diagnosis, preimplantation genetic diagnosis, single nucleotide genotyping, epidemiology studies, forensic biology and sample archiving. However, it is apparent from the literature that isothermal WGA methods, specifically MDA, may be on the verge of replacing older PCR-based
WGA techniques. By performing an isothermal reaction at a constant temperature, MDA avoids sequence-specific factors such as GC content, secondary structure and template length that may cause preferential amplification of specific regions during PCR thermalcycling. φ29 DNA polymerase has approximately 10 times greater replication fidelity compared to Taq DNA polymerase (Esteban et al., 1993). The high
46 processivity and strand-displacing activity of φ29 DNA polymerase ensure faithful replication of template DNA, in contrast to PCR-based methods that exhibit incomplete loci coverage and thus generate amplified product that do not reliably represent the original template.
Hence, MDA products exhibit a maximum amplification bias of 3-fold, compared to bias of up to 1,000,000-fold inherent in PCR-based WGA products. In addition, PCR-based WGA generates products less than 2kb in length, significantly shorter than the 70kb fragments that can be generated by MDA.
Although the level of amplification bias evident in MDA products is relatively low, the use of MDA products may still pose a problem where allelic copy numbers are under analysis and any bias greater than 2-fold cannot be tolerated. This is often the case in mixture analysis of forensic crime scene samples that may contain DNA from two or more contributors, and also in comparative genomic hybridisation where allelic copy number changes in tumour samples or preimplantation genetic diagnosis of viable IVF embryos are under examination. As yet there exists no reported method that offers to overcome amplification bias in MDA products. Hence, the effective use of MDA in many diagnostic applications remains to be achieved.
The research outlined in this thesis arose out of the perceived need to provide a better WGA technique that generates more representative
DNA from limiting DNA samples that can be utilised in a variety of
47 specialist areas, namely forensic investigations and clinical disease diagnostics. Due to its characteristics that overshadow those of other
WGA techniques, MDA by φ29 DNA polymerase was selected as the focus of this research. Specifically, the commercially-available
GenomiPhi™ DNA Amplification Kit from Amersham Biosciences
(Piscataway, NJ, USA; now part of GE Healthcare) was used as the
MDA system of choice. Although other commercial MDA systems such as REPLI-g™ from Molecular Staging (New Haven, CT, USA; now part of Qiagen GmbH) were available at the time, the decision to use
GenomiPhi™ was primarily due to the fact that the Cooperative
Research Centre for Diagnostics, Queensland University of Technology, was previously selected as a beta test site for the product and in-house experience with the GenomiPhi™ system was available.
1.5. Hypothesis and Research Aims
1.5.1. Hypothesis
Multiple Displacement Amplification (MDA) can be used for the representative amplification of a variety of human biological, forensic and pathology samples at levels down to a single copy genome, allowing effective diagnostics from precious and/or limited samples.
48 1.5.2. Research Aim I
Aim I: to implement an MDA procedure on a range of geographically diverse population samples of limited availability to create an archive of these samples.
Aim I assessed the use of MDA as a sample archiving tool for precious or limited DNA samples. This aim included an evaluation of MDA sensitivity to low quantities of template DNA and examined the compatibility of the 5% Chelex extraction procedure with the standard
MDA protocol.
1.5.3. Research Aim II
Aim II: the utility of MDA for the amplification of a range of simulated forensic samples containing low levels of DNA, such as saliva stains
(cigarette butts, licked stamps and envelopes), fingernail scrapings, touched objects, vaginal swabs, semen, shed hairs, and bone/skeletal remains.
This research aim examined the effects of degraded DNA as templates for MDA, and the use of MDA as a tool to amplify low quantities of
DNA extracted from simulated forensic samples.
49 1.5.4. Research Aim III
Aim III: to establish an MDA protocol for the amplification of single cells for use in comparative genomic hybridisation (CGH) and preimplantation genetic diagnosis (PGD) from a variety of pathology samples, including embryonic blastomeres, amniocytes and tissue biopsy material.
1.6. Research Design
The yields and performance of MDA-amplified DNA was compared to the original unamplified DNA template by any of the following assays: single locus PCR, quantitative real-time PCR, or comparative genomic hybridisation (CGH). A modified MDA protocol that reduces amplification bias was also compared to the original unamplified DNA template using similar assays.
1.7. Thesis Outline
This section provides a brief outline of this thesis for the benefit of the reader.
50 Chapter Two provides an in-depth description of the materials and methods used throughout the investigation. Protocols for sample collection and a variety of extraction procedures, including techniques that aim to reduce contamination are provided. Other protocols that are described include methods for quantitation; quantitative real-time PCR; cycling conditions for the amplification of specific SNP-containing fragments by PCR; agarose gel electrophoresis; the standard
GenomiPhi™ MDA method and a modified GenomiPhi™ method; the controlled fragmentation of genomic DNA to create artificially-degraded
DNA; and comparative genomic hybridisation (CGH).
Chapter Three covers Research Aims I and II. The first half of the chapter primarily concerns Aim II. This section consists of an introduction to the problems faced in the field of forensic biology concerning samples of low copy number DNA. The reader is introduced to the variety of sample types that are commonly encountered in the field of forensic biology. The sensitivity of MDA to a dilution series consisting of genomic DNA or individual human cells provides an appreciation of the utility of MDA for the amplification of samples containing low copy number DNA. Two extraction methods, 5% Chelex and buffer digest with Proteinase K, are assessed for compatibility with the MDA protocol, and issues pertaining to reaction inhibition are addressed in this section. Simulated forensic samples are used to determine utility of MDA in forensics. The use of MDA products is demonstrated for a variety of assays, such as PCR and real-time
51 quantitative PCR. Furthermore, the effect of degraded DNA templates on MDA reaction kinetics is presented. The second half of the chapter concerns the archiving of precious DNA samples, and yields for MDA of limited bloodspots are presented. Theorised yields of scalable MDA reactions are given. Future directions for utility of MDA in forensic biology are offered.
In Chapter Four, the use of samples containing low quantities of DNA, specifically single cells such as amniocytes, embryonic blastomeres and tissue biopsy material for the purposes of clinical diagnostics is briefly described. The reader is offered an appreciation of the state of technology that is available for use in disease analysis, and new developments that have arisen as a result of limitations imposed on the techniques due to limited sample availability. The problem of 3-fold amplification bias in MDA-amplified DNA is examined, and a modified
MDA method is proposed as a possible solution to overcome problems of amplification bias. The effect of hybridising high molecular weight
DNA from MDA primer concatemers to human metaphase spreads was analysed by CGH in order to determine the possibility of misdiagnosis due to amplified random primer complexes. Future directions for MDA in clinical diagnostics are offered.
Chapter Five provides a final summary and conclusion. The relevance of the research results to the field of forensic biology and clinical diagnostics are highlighted. This chapter also describes future research
52 into the field of DNA amplification technology that could be performed to build on the outcomes of this thesis.
1.8. Conclusion
In this chapter, I presented the problem, the rationale behind the investigation, the hypothesis, research aims, and the research design.
Now that the reader understands the importance of the research undertaken and how I have presented it in this thesis, the reader can continue by going into the main body of the thesis.
53 CHAPTER TWO
MATERIALS AND METHODS
2.1. Introduction
This chapter describes in detail the materials and methods used throughout the investigation. The different techniques are divided into six sections: (1) sample handling and DNA preparation; (2) DNA extraction and quantification; (3) DNA amplification and agarose gel electrophoresis; (4) multiple displacement amplification (MDA); (5) artificial degradation of DNA; and (6) comparative genomic hybridisation (CGH).
2.2. Sample Handling and Preparation of DNA Samples
2.2.1. General sample handling
Samples were handled using protocols that are similar to those utilised in molecular archaeology for DNA analysis of precious or ancient samples
(O’Rourke et al., 2000). These protocols aim to reduce the possibility of
54 environmental contamination and the occurrence of cross-contamination between items, thus ensuring the authenticity of the DNA under analysis.
All DNA analyses (handling, extraction, addition of template DNA for
PCR) were performed in a Class II Biohazard Hood, sterilised with 10% bleach (sodium hypochlorite solution), 70% ethanol and UV-irradiated for a minimum of 1 hour before use. Only one type of analysis (either handling, extraction or addition of template DNA for PCR) was performed at any one time. The Class II Biohazard Hood was completely decontaminated and re-sterilised after the completion of each procedure.
All tools required for any type of analysis (eg. pipettors, boxes of pipette tips, stainless steel scissors, scalpels, scalpel blades, tweezers, plastic tube racks, container of reaction tubes, marker pens, beakers, etc) were wiped with 10% bleach and 70% ethanol before exposing each side of the tool to UV-irradiation for a minimum of 30 minutes. Cutting implements were re-sterilised with 10% bleach and 70% ethanol between each use.
Double-distilled water (ddH2O) was used to prepare all solutions.
Dilutions of DNA standards and DNA extracts were made with sterile
ET buffer (0.1mM EDTA, 0.1mM Tris-HCl, pH 7.5) or nuclease-free injectable H2O (Pharmacia & Upjohn, Kalamazoo, MI, USA). Purified amplification products for use in comparative genomic hybridisation
55 (CGH) were eluted in nuclease-free injectable H2O (Pharmacia &
Upjohn, Kalamazoo, MI, USA).
Gloves were changed between analysing different samples to prevent cross contamination. Negative controls consisting of sterile ddH2O in place of DNA extract were included in all sample-handling procedures to monitor the occurrence of contamination. Positive controls, containing a sample or DNA extract of known quality and reliability, were also included in all tests to assess the performance of the extraction, quantification or amplification procedures.
2.2.2. Dilution of human genomic DNA for sensitivity assays
Commercially quantified normal human male genomic DNA was purchased from Promega (Promega Corp., Madison, WI, USA) and diluted by 10-fold serial dilutions into a series containing 1 to 1,000 genomic equivalents (GEq) per microliter. One GEq represents 6.4pg of genomic DNA, the amount of DNA present within a single human male diploid cell (Dolezel et al., 2003). The dilutions were prepared in ET buffer (0.1mM EDTA, 0.1mM Tris-HCl, pH 7.5).
2.2.3. Collection of human fibroblasts for sensitivity assays
Karyotypically-normal human male fibroblasts were donated by the
Genetics Department, Queensland Medical Laboratory, West End,
56 Brisbane QLD Australia. Cells were suspended in phosphate buffered saline (PBS pH 7.5) and were subjected to multiple displacement amplification (MDA) without prior purification or lysis procedures.
2.2.4. Collection and preparation of simulated forensic specimens
(buccal swabs, licked stamps, touched object samples and shed hairs)
All samples for simulated forensic specimens were obtained from laboratory colleagues with informed consent and within the guidelines and approval of the Queensland University of Technology Human
Resources Ethics Committee. Buccal swabs were prepared by sampling the inside of the cheeks with a sterile cotton swab (Copan Italia S.P.A.,
Brescia, Italy). The swab was air-dried and half of the swab was cut and added to a 0.5mL tube. Saliva samples were collected from freshly- licked 90c Australia Post postage stamps. After air-drying, portions
(5mm x 5mm squares) of each licked stamp were sampled into a 0.5mL tube. For touched objects, donors were each given a sterilised Staedtler
Stick 430 M pen (Staedtler, Dee Why, NSW, Australia) and asked to copy a length of predetermined text using the supplied pen. Sterile cotton swabs (Copan Italia S.P.A., Brescia, Italy) moistened with distilled H2O were used to swab the pen shafts. After air-drying, portions of the swab were cut using sterilised scissors and added to a 0.5mL tube.
Shed hairs were recovered from unshared items such as clothing, bedding, hair-brushes and placed in individual sealable Ziploc plastic bags for storage at room temperature.
57 2.3. DNA Extraction and Quantification
2.3.1. DNA extraction of population database bloodspots by 5% Chelex- 100
Extraction of dried bloodspots by 5% Chelex-100 (herein referred to as
5% Chelex) was performed as described previously (Walsh et al., 1991), with slight modifications. Briefly, a 5mm bloodspot was punched using a sterilised hole-puncher and added to a 0.5mL tube, followed by the addition of 400µL ET buffer (0.1mM EDTA, 0.1mM Tris-HCl, pH 7.5).
The tube was vortexed for 5 seconds and incubated at room temperature on an automatic shaker (Eppendorf, Hamburg, Germany) for 30 minutes, before vortexing for 5 seconds and spinning in a tabletop microcentrifuge at maximum speed (13,000rpm) for 1 minute. The supernatant was carefully discarded, leaving sufficient liquid to immerse the matrix and pellet. Using a wide-bore pipette tip, 200µL of evenly mixed 5% (w/v) Chelex-100 solution (Sigma-Aldrich, Sydney, NSW,
Australia) was added to the pelleted sample and the sample was incubated at 56°C for 30 minutes. The tube was subsequently vortexed for 10 seconds and incubated at 99°C for a further 8 minutes. After vortexing for another 10 seconds, the tube was centrifuged at maximum rpm for 3 minutes.
58 2.3.2. DNA extraction of simulated low copy number forensic samples
(buccal swabs, licked stamps, touched object samples) by a modified 5%
Chelex protocol
Extraction of simulated forensic samples (buccal swabs, licked stamps and touched objects) was performed using a 5% Chelex-100 method as described previously (Walsh et al., 1991), with slight modifications. To the tubes containing prepared samples, 50µL of a 5% (w/v) Chelex-100 suspension (Sigma-Aldrich, Sydney, NSW, Australia) was added using wide-bore pipette tips. The mixture was incubated at 56°C for 30 minutes, vortexed for 10 seconds, heated at 99°C for 8 minutes and vortexed again, before centrifuging the extract on a tabletop microcentrifuge at maximum speed (13,000rpm) for 3 minutes.
2.3.3. Extraction of DNA from shed hairs by a modified
Buffer/Proteinase K digest protocol
DNA from shed hair samples was extracted using a buffer digest protocol based on a method described previously (Allen et al., 1998).
Briefly, using sterile tweezers and scissors, a 4cm portion of shed hair containing the follicle was cut and vigorously washed in sterile ddH2O for 2 minutes before washing vigorously in 100% ethanol for 2 minutes.
The piece of shed hair was air-dried on a piece of clean blotting paper for 3 minutes, then cut into 5mm portions and placed in a 0.5mL microcentrifuge tube containing 20µL of 1x Platinum® Taq PCR buffer
59 (20mM Tris-HCl pH 8.4, 50mM KCl) (Invitrogen Corp., Carlsbad, CA,
USA) and 2µL of Proteinase K (20mg/mL). The tube was centrifuged briefly and incubated at 56°C for 3 hours. The Proteinase K was inactivated at 95°C for 10 minutes.
2.3.4. Procedure for salt extraction of genomic DNA from whole blood for use in Comparative Genomic Hybridisation (CGH)
DNA from blood was isolated using a salt extraction procedure. Briefly,
TE buffer (10mM Tris-HCl, 0.1mM EDTA, pH 8.0) was added to 6mL of blood, mixed and centrifuged at 686g for 10 minutes at 4°C.
Following removal of the supernatant, the pellet was resuspended in
1mL of lysis buffer (100 µM Tris, 2mM EDTA, 100 µM NaCl) and mixed well. 360µg of Proteinase K, 35µL of 20% SDS and 40µg of
RNase A (Roche Applied Science, Indianapolis, IN, USA) were added and mixed before centrifuging at 19800g for 15 minutes at 4°C. The supernatant was transferred to a clean tube before adding 2.5 volumes of cold 100% ethanol to precipitate the DNA. DNA was pelleted with a
1240g spin for 5 minutes. The DNA pellet was washed with 1mL of
70% ethanol, which was then removed and the DNA left to dry at room temperature.
The DNA pellet was resuspended in TE buffer and allowed to equilibrate at 4°C for two days, before quantitation by spectrophotometric
60 260nm/280nm absorbance (Section 2.3.6) and dilution of the DNA to
1ng/µL in ET buffer.
2.3.5. Quantification of samples by quantitative real-time PCR (RT-
QPCR)
The quantitative real-time PCR mix consisted of 1x SYBR Green reagent (Applied Biosystems, Foster City, CA, USA), 0.4µM primer set
(GeneWorks, Hindmarsh, SA, Australia) and 2µL of DNA template in a total reaction mix of 20µL, with samples analysed in duplicates. The standard curve was composed of a 10-fold dilution series of unamplified genomic DNA (as described in section 2.2.2.). Samples were amplified on an ABI Prism 7000 SDS (Applied Biosystems, Foster City, CA,
USA), with a thermal cycling program consisting of a 1 minute incubation at 50°C for UDG digestion, Taq activation and DNA denaturation at 95°C for 10 minutes, followed by 35-40 cycles of denaturation at 94°C for 15 seconds, a primer-specific annealing step
(Table 2.1) for 20 seconds and detection at 72°C for 33 seconds. The block was cooled to 50°C for 1 minute before ramping a dissociation step from 60-95°C to determine amplification and detection of the correct target. The quantitation was only accepted if the correlation co- efficient (r2) of the standard curve was greater than 98%.
61 Table32.1. Primer sequences for quantitative real-time PCR amplification on the ABI 7000 SDS platform.
Target Primer Sequence Tm Fragment (°C) size (bp) TYRP1 F: 5’-GTATCCCCATGATGGCAGAG-3’ 61 102 R: 5’-GTCCCACAGTTGTGTCCTGA-3’ RABGGT F: 5’-CAGTACTTCCAGACCCTCAAGGCAAG-3’ 61 103 R:5’-GAGGTGGGCAGGGTATGCATGTCAGC-3’ mtDNAg1719a F: 5’-TAAACCTAGCCCCAAACCCA-3’ 55 112 R: 5’-TACTATATCTATTGCGCCAGG-3’
2.3.6. Quantification of whole blood genomic DNA and MDA products by spectrophotometric 260nm/280nm absorbance
Genomic DNA extracted from the whole blood sample (Section 2.3.4) was diluted by 5:100 in ET buffer (0.1mM EDTA, 0.1mM Tris-HCl, pH
7.5) prior to quantitation by UV 260nm/280nm absorbance spectrophotometry.
For quantitation of MDA products, the reactions were purified by ethanol precipitation as per the GenomiPhi™ DNA Amplification Kit user’s protocol and resuspended in 50µL sterile injectable H2O
(Pharmacia & Upjohn, Kalamazoo, MI, USA). The concentration of
DNA was determined from a 3:100 dilution of the purified MDA product by 260nm/280nm absorbance, with 320nm background correction, using a DU®600 or DU®800 UV/Visible spectrophotometer
(Beckman Coulter, Fullerton, CA, USA). Multiple readings were taken, and an average concentration was calculated.
62 The sample blanks used to calibrate the spectrophotometers consisted of
100µL of sterile injectable H2O (Pharmacia & Upjohn, Kalamazoo, MI,
USA) or ET buffer, where appropriate. A control sample containing
145ng/µL human genomic DNA (Promega Corp., Madison, WI, USA) was quantified alongside the samples, and measurements were only accepted if the values for this control sample were within 5% of
145ng/µL.
2.4. PCR Amplification and Agarose Gel Electrophoresis
PCR reactions for five unique human genomic DNA regions and two human mitochondrial DNA regions were carried out using oligonucleotide primers as described in Table 2.2. Oligonucleotide primers were designed using either Primer3 v0.2
(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) or Primer
Express® v2.0 (Applied Biosystems, Foster City, CA, USA). Genomic
DNA primers were manufactured by GeneWorks (GeneWorks,
Hindmarsh, SA, Australia), and mitochondrial DNA primers were manufactured by Proligo (Proligo Australia, Lismore, NSW, Australia).
® PCR buffer, MgCl2 and Platinum Taq polymerase were obtained from
Invitrogen (Invitrogen Corp., Carlsbad, CA, USA). PCR reactions consisted of 1x Platinum® Taq PCR Buffer (20mM Tris-HCl pH 8.4,
® 50mM KCl), 1.5mM MgCl2, 0.02U/µL Platinum Taq DNA polymerase, 0.8mM total dNTPs (Roche Applied Science, Indianapolis,
63 IN, USA), 0.5µM primers and 1µL DNA template. DNA template was added to the PCR mastermix in a Class II Biohazard Hood, separated from areas where PCR products are under analysis. ET buffer was added for a total PCR reaction volume of 25µl. PCR amplification was performed on a PTC-200® Peltier Thermal Cycler (MJ Research,
Waltham, MA, USA), with thermocycling protocols as outlined in Table
2.2. PCR positive and negative controls consisted of 50 genomic equivalents (320pg normal human male genomic DNA) and 1µL ET buffer respectively.
Fragment analysis of PCR products was performed by electrophoresis on a 1.5% agarose gel using 0.5x TAE buffer (Tris-Acetate-EDTA) at 110V for 30 minutes. The gel was stained by ethidium bromide and viewed under UV illumination.
64 Table42.2. Nuclear and mitochondrial DNA targets and thermocycling conditions used for PCR amplification.
Human Target Primer Sequences Initial PCR Cycling Protocol* Fragment Genome Denaturation size (bp)
(°C x mins) Denaturation Primer Tm Extension (°C x mins) (°C x mins) (°C x mins)
F: 5’-TTCTCGGGCGGGTGATCCCT-3’ Agouti 55°C x 0:20 100 R: 5’-AAGCCAGGTCTCCCTGAAGT-3’ F: 5’-GTATCCCCATGATGGCAGAG-3’ TYRP1 60°C x 0:20 102 A R: 5’-GTCCCACAGTTGTGTCCTGA-3’ N D
F: 5’-CCAAAGCAAGAAGTCAGCT-3’ r
a OA1 94°C x 2:00 94°C x 0:20 53°C x 0:20 72°C x 0:20 233
e R: 5’-GCGATTTGAGGAGCATAAGTA-3’ l
uc F: 5’-CCTACTCCAACCTTGCGGTGAAAGATCG-3’
N CITED1 65°C x 0:20 380 R: 5’-TTCCTCATCCACTGGGTCCGAATCGATG-3’ F: 5’-CCCCAGAGAAGGACAAATTTTTTGCCTACC-3’ TYR 65°C x 0:20 497 R: 5’-TGAAGAAGTGATTGTTAAGGTTCCTCCCTACTC-3’
F: 5’-TAAACCTAGCCCCAAACCCA-3’
A g1719a 55°C x 1:00 112 R: 5’-TACTATATCTATTGCGCCAGG-3’ N 95°C x 2:00 95°C x 0:20 72°C x 0:40 D
t F: 5’-CCTCACCCACTAGGATACCA-3’
m g16391a 55°C x 1:00 194 R: 5’-GGAGCGAGGAGAGTAGCAC-3’ *Number of cycles: 34 cycles for nuclear DNA targets and 40 cycles for mtDNA targets.
65 2.5. Multiple Displacement Amplification (MDA)
2.5.1. Multiple Displacement Amplification (MDA) using the
GenomiPhi™ DNA Amplification Kit
Multiple Displacement Amplification (MDA) by φ29 DNA polymerase was performed using the GenomiPhi™ DNA Amplification Kit
(Amersham Biosciences, Piscataway, NJ, USA), according to the manufacturer’s instructions. Briefly, in 0.2mL reaction tubes, 1µL of
DNA template was added to 9µL of GenomiPhi™ Sample Buffer. The sample mix was denatured at 95°C for 3 minutes, and then cooled on ice for 5 minutes before centrifuging to collect any condensation. To the denatured sample, a mix containing 9µL of GenomiPhi™ Reaction
Buffer and 1µL of GenomiPhi™ Enzyme Mix was added and mixed gently. The reaction mix was incubated at 30° for 18 hours. After amplification, the enzyme was inactivated at 65°C for 10 minutes.
After completion of the MDA reaction, 2µL of GenomiPhi™ MDA product diluted 1:2 with sterile injectable H2O (Pharmacia & Upjohn,
Kalamazoo, MI, USA) was electrophoresed against uncut λ DNA on a
1.5% agarose gel using 0.5x TAE buffer (Tris-Acetate-EDTA) at 100V for 30 minutes, stained by ethidium bromide and viewed under UV illumination.
66 2.5.2. A Modified MDA procedure using the GenomiPhi™ DNA
Amplification Kit
A novel modification of the GenomiPhi™ protocol was developed.
Briefly, in a 0.2mL reaction tube, 1µL of DNA template was added to
9µL of GenomiPhi™ Sample Buffer. The sample mixture was divided into two portions of 5µL each: one remained non-denatured and the other was exposed to denaturation at 95°C for 3 minutes and then cooled on ice for 5 minutes before a brief centrifugation to collect any condensation. The two 5µL portions were mixed, and a solution containing 9µL of GenomiPhi™ Reaction Buffer and 1µL of
GenomiPhi™ Enzyme Mix was added and mixed gently. The reaction mix was then incubated at 30° for 18 hours. After amplification, the enzyme was inactivated at 65°C for 10 minutes.
After completion of the MDA reaction, 2µL of GenomiPhi™ MDA product diluted 1:2 with sterile injectable H2O (Pharmacia & Upjohn,
Kalamazoo, MI, USA) was electrophoresed against uncut λ DNA on a
1.5% agarose gel using 0.5x TAE buffer (Tris-Acetate-EDTA) at 100V for 30 minutes, stained by ethidium bromide and viewed under UV illumination.
67 2.6. Artificial Degradation of Genomic DNA by Bovine Pancreatic
DNase I
Artificial degradation of human genomic DNA was performed using a two-fold titration of bovine pancreatic DNase I enzyme, based on a method published previously (Bender et al., 2004). All DNase reaction components, including human male genomic DNA, were obtained from
Promega (Promega Corp., Madison, WI, USA). Reactions were prepared on ice. The final reaction components in each tube contained 1x RQ1
RNase-Free DNase Reaction Buffer (40mM Tris-HCl pH 8.0, 10mM
MgSO4, 1mM CaCl2), 145ng of human male genomic DNA, one of the following concentrations of RQ1 RNase-Free DNase enzyme: 0.1U,
0.05U, 0.025U, 0.0125U, 0.00625U, and TE buffer (10mM Tris-HCl,
0.1mM EDTA, pH 8.0) to a final volume of 10µL. Reactions were incubated at 37°C for 5 minutes and inactivated by adding 1µL of RQ1
RNase-Free DNase Stop Solution (20mM EGTA, pH 8.0) with further incubation at 65°C for 10 minutes.
Fragment sizes of degraded DNA samples were determined by electrophoresis of 4µL digested DNA on a 1% agarose gel using 0.5x
TAE (Tris-Acetate-EDTA) at 110V for 30 minutes. The gel was stained by ethidium bromide and viewed under UV illumination. Samples were purified by ethanol precipitation and resuspended in 50µL of ET buffer.
Fragment sizes were confirmed via PCR amplification of the purified samples using different amplicon lengths. For MDA, 1µL of the purified
68 sample was added to the MDA reaction. Following MDA, the reaction products were diluted to 1:10. 1:100, 1:1,000 and 1:10,000 for PCR amplification using TYRP1 and mtDNAg1719a primer sets.
2.7. Comparative Genomic Hybridisation (CGH)
GenomiPhi™ reaction products were purified by ethanol precipitation as per the GenomiPhi™ DNA Amplification Kit protocol and resuspended in 50µl of sterile injectable H2O (Pharmacia & Upjohn, Kalamazoo, MI,
USA). The DNA concentration was determined by spectrophotometric quantification (as described in section 2.3.6.), and 1µg used for CGH.
CGH analysis was performed using Vysis Comparative Genomic
Hybridisation Reagents (Vysis, Downers Grove, IL, USA), according to the manufacturer’s instructions with some modifications. Nick translation of the GenomiPhi™ was performed to generate probes labelled with the SpectrumGreen™ fluorophore (Vysis, Downers Grove,
IL, USA). The nick translation reaction was assembled by mixing 1µg of either unamplified normal human male genomic DNA or GenomiPhi™- amplified product along with 5µL of 0.1mM dATP, dCTP, dGTP mix,
1µL of 0.1mM dTTP, 1µL of 0.2mM SpectrumGreen™ dUTP, 5µL of
Nick Translation Enzyme Mix (Vysis, Downers Grove, IL, USA), 1µL of DNA Polymerase I 10U/µL (Invitrogen Corp., Carlsbad, CA, USA), and injectable nuclease free H2O (Pharmacia & Upjohn, Kalamazoo, MI,
69 USA) to a total volume of 50µl. The mix was incubated at 15°C for 40-
60 minutes to generate sufficient probes of length between 300-1000bp, determined by electrophoresis against an appropriate DNA size standard on a 1% agarose gel with 0.5x TBE (Tris-Borate-EDTA) at 117V for 30 minutes. Nicking enzymes were inactivated at 75°C for 10 minutes, and the reaction was purified using a Sephadex G-50 Quick Spin column
(Roche Applied Science, Indianapolis, IN, USA) as per the manufacturer’s instructions.
Purified probes labelled with SpectrumGreen™ were mixed with an equal amount of SpectrumRed™-labelled Normal Male Reference DNA
(Vysis, Downers Grove, IL, USA) and ethanol-precipitated overnight at
-70°C in the presence of 20µL of Human Cot1 DNA (1mg/mL)
(Invitrogen Corp., Carlsbad, CA, USA). Probes were pelleted by centrifugation at 14,000rpm for 30 minutes at 4°C, the supernatant removed and the pellet allowed to completely dry in the dark. The pellet was resuspended in 10µl Hybridisation Mix (Vysis, Downers Grove, IL,
USA) and denatured at 74°C for 6 minutes. Denatured probes were added to a microscope slide containing denatured metaphases from a normal human subject, covered with a coverslip and sealed under rubber cement. The slide was placed in a humidified chamber and incubated at
37°C for 65 hours.
After incubation, the metaphase slide was washed in a solution of 50% formamide, 2x saline sodium citrate pH 7.0 at 43°C for 5 minutes with
70 frequent agitation. The slide was then washed in phosphate-buffered saline (PBS) pH 7.25 for 1 minute, counterstained in 4’,6-Diamidino-2- phenylindole (DAPI) for 2 minutes with a brief rinse in PBS pH 7.25.
Vectashield Mounting Medium for Fluorescence (Vector Laboratories
Inc., CA, USA) was added to the slide prior to applying a coverslip.
Visualisation was performed on a Leica DM LB fluorescence microscope with an attached CCD camera and computer installed with the CytoVision version 2.77 (Applied Imaging, San Jose, CA, USA) analysis software.
2.8. Conclusion
The materials and methods used for sampling handling, DNA preparation, DNA extraction and quantification, PCR amplification and analysis, MDA and CGH were described in this chapter. Results from these experiments are presented in the next two chapters.
71 CHAPTER THREE
APPLICATION OF MULTIPLE DISPLACEMENT
AMPLIFICATION IN FORENSIC BIOLOGY
3.1. Introduction
At the most basic level, the isolation of sufficient DNA is a fundamental requirement for any molecular biology analysis. However, there are many fields of DNA testing, including forensic biology, clinical diagnostics, and molecular archaeology where amounts of sample are often very limiting. Furthermore, there is an increasing demand in the field of forensic biology to analyse every piece of evidence for trace
DNA deposits. Examples of crime scene exhibits that may contain a limited amount of DNA include cigarette butts and saliva stains (Walsh et al., 1992; Sweet et al., 1996), shed hairs (Takayanagi et al., 2003), degraded remains (Evison et al., 1997), ejaculate material (Walsh et al.,
1991) and also touched objects or fingerprints (van Oorschot and Jones,
1997; van Oorschot et al., 2003; Lowe et al., 2002). Trace samples often yield very little DNA and therefore the whole sample may be quickly consumed by one or two PCR reactions, preventing confirmatory testing
72 or high-throughput downstream analysis. Bodily fluids such as bloodstains and semen stains may contain a higher concentration of nucleated cells and therefore yield more DNA. However, evidence material such as these may also be limiting due to sample size. Current forensic DNA testing by short tandem repeat (STR) PCR requires approximately 1 to 2.5ng of DNA to ensure optimum amplification with the widely used AmpFlSTR® Profiler Plus® kit (Applied Biosystems
AmpFlSTR® Profiler Plus® PCR Amplification Kit User’s Manual,
1998). However, many samples collected from crime scenes do not contain this amount of DNA and may fail to yield an informative STR profile.
Whole genome amplification (WGA) methods have been developed to amplify the DNA in limiting samples into adequate amounts for thorough analysis. As outlined in Chapter I, most WGA methods employ protocols that are based on PCR, such as primer extension preamplification (PEP) (Zhang et al., 1992) and degenerate oligonucleotide-primed PCR (DOP-PCR) (Telenius et al., 1992).
Unfortunately, PCR-based WGA methods generate short (200-1000bp) products and do not provide complete coverage of the genome, resulting in amplification bias (Zhang et al., 1992; Cheung and Nelson, 1996). In addition, the effect of any slippage errors can be multiplied in subsequent amplification cycles during PCR WGA (Hawkins et al.,
2002). As PCR-based WGA methods are prone to high levels of amplification bias and error, the utility of these WGA methods in the
73 field of forensic biology, specifically for the identification of suspects in criminal investigations or the identification of deceased individuals in mass disaster victim identification (DVI) cases, has not been routinely reported due to the high chances of misidentification.
The recently introduced isothermal WGA method termed multiple displacement amplification (MDA) opens the possibility for the use of
WGA in forensic biology. A detailed outline of the MDA procedure was presented in Chapter I. Briefly, MDA is based on the strand-displacing properties of φ29 DNA polymerase in rolling circle amplification (Dean et al., 2001; Dean et al., 2002). The polymerase carries out a highly processive and continuous elongation of each DNA strand without dissociating from the template, synthesising long fragments up to 70kb during a short isothermic incubation period (Blanco et al., 1989). φ29
DNA polymerase exhibits 3’→5’ exonuclease proofreading activity and a low error rate that ensures uniform amplification of DNA template with minimal amplification bias (Esteban et al., 1993).
MDA products have been shown to provide similar results to their original genomic DNA templates when comparisons were made with
SNP, TaqMan, RFLP, and whole genome hybridisation assays in addition to STR profiling and direct sequencing (Dean et al., 2002;
Hosono et al., 2003; Tranah et al., 2003; Paez et al., 2003). MDA results in efficient amplification of a wide range of sample types and is highly sensitive to samples containing very small concentrations of DNA (Dean
74 et al., 2002). It was shown that MDA is capable of producing consistent yields of up to 30µg from amounts of input DNA ranging from 10ng to
100fg (Dean et al., 2002). The characteristics of the MDA reaction and the consistency of the product yield make it ideal for routine use in the field forensic DNA testing.
The utility of MDA in forensic biology has not been assessed, except in a small study performed recently by Sorensen et al. (2004), who focused on amplifying buccal swabs and simulated latent fingerprints. Their findings showed that MDA products allowed a much greater frequency of successful STR typing using the GammaSTR™ multiplex STR kit
(Promega Corp., Madison, WI, USA) compared to unamplified samples;
60% and 37.5% success rate for MDA of buccal swabs and fingerprints respectively, compared to 30% and 0% for buccal swabs and fingerprints respectively without MDA amplification (Sorensen et al., 2004). MDA was able to successfully amplify down to 8-10 single human lymphoblastoid cells, generating sufficient DNA for more than 5,000 alu-PCR reactions (Sorensen et al., 2004). MDA was performed using the REPLI-g™ system (Molecular Staging, New Haven, CT, USA; now part of Qiagen GmbH).
Another small study was performed by Schneider et al. (2004), who assessed the utility of MDA-amplified human cells for use in STR typing. Dilutions down to 5pg, containing human cell lines HepG2 and
P118, were amplified by MDA prior to amplification using the
75 PowerPlex® 16 (Promega Corp., Madison, WI, USA) and AmpFlSTR®
SGM Plus® (Applied Biosystems, Foster City, CA, USA) multiplex STR kits. MDA was performed using both the REPLI-g™ (Molecular
Staging, New Haven, CT, USA) and GenomiPhi™ (Amersham
Biosciences, Piscataway, NJ, USA) systems. Results showed reliable
STR typing from MDA products generated using 500pg of genomic
DNA, the equivalent of approximately 70 single human genomes
(Schneider et al., 2004). Allelic dropouts started to occur at 50pg, with increased dropouts at 5pg, and no difference between the two STR kits were noticeable once the genomic DNA was amplified (Schneider et al.,
2004). However, the REPLI-g™ system was found to generate approximately double the amount of product compared to GenomiPhi™
(Schneider et al., 2004).
As yet, there has not been a lot of work invested in assessing the use of
MDA for the amplification of low copy number DNA samples that are routinely encountered in the field of forensic biology, such as saliva- stained material (cigarette butts, licked stamps), single shed hairs, touched objects and degraded DNA specimens that may be encountered in disaster victim identification cases. The compatibility of MDA to
DNA samples extracted using common procedures such as Chelex-100 has not been assessed. A thorough analysis on the utility of MDA as a tool for generating DNA archives of precious or limited samples is also absent from the literature.
76 This chapter presents the research outcomes of Aims I and II. Aim I included the development of an MDA procedure on a range of geographically diverse population samples of limited availability, in the form of single bloodspots on cotton cloth or paper matrices, to create an archive of these samples. Aim II involved assessing the utility of MDA for the amplification of a range of simulated forensic samples containing low levels of DNA, such as saliva stains (cigarette butts, licked stamps and envelopes), fingernail scrapings, touched objects, vaginal swabs, semen, shed hairs, and bone/skeletal remains. However, due to the limited availability of the bloodspot samples, Aim II was performed prior to Aim I because a larger number of samples were available for use. This was especially important as it allowed the optimisation of extraction procedures to ensure maximum compatibility with the MDA procedure.
Results in this chapter include an assessment on the sensitivity of MDA to a genomic DNA dilution series and a dilution of individual human fibroblasts, down to the single genome level. Simulated forensic specimens containing limited amounts of DNA were used to assess the utility of MDA for application in the field of forensic biology. Two common DNA extraction methods, namely extraction by 5% Chelex and buffer digest with Proteinase K, were examined for compatibility with the MDA protocol. High molecular weight genomic DNA was also artificially degraded to observe the effect of degraded DNA templates on
MDA reaction kinetics. Archiving of precious DNA samples, present in
77 the form of single bloodspots, was performed using MDA. A discussion is included as each result is presented in order to help the understanding of the developments that were made as the project proceeded. The chapter finishes with a conclusion that ties together the findings for both aims. Future directions for the utility of MDA in forensic biology are also offered.
Portions of the work outlined in this chapter were presented at the 7th
International Conference on Ancient DNA and Associated Biomolecules
(Brisbane, QLD Australia; 10-17 July 2004), and the 15th International
Symposium on Human Identification (Phoenix, AZ USA; 4-7 October
2004). Part of this work has been submitted as a Technical Note to the
Journal of Forensic Sciences on the 14th of July, 2005.
3.2. Results and Discussion
3.2.1. Sensitivity and yield of MDA on low DNA concentrations down to one single human cell
MDA using the GenomiPhi™ DNA Amplification Kit (Amersham
Biosciences, Piscataway, NJ, USA) generated DNA product from samples containing low levels of DNA. Analysis of a dilution series containing 6.4pg to 6.4ng of human genomic DNA, or 1 to 1,000 human genome equivalents, showed that the MDA reaction amplified all sample
78 concentrations (Figure 3.1.A). This sensitivity was confirmed with MDA of a cell dilution series consisting of 1, 2, 5 and 10 individual human male fibroblasts (Figure 3.1.B). In both cases, all MDA reactions generated high molecular weight DNA product that could be further amplified by PCR, and amplicons were observed for the whole range of
DNA dilutions examined.
Figure43.1. Agarose gel analysis of MDA and PCR reactions from genomic DNA or single cell dilutions. (A) Agarose gel analysis of MDA and PCR reactions of a genomic DNA dilution series, consisting of normal human male genomic DNA ranging from 1000 genomic equivalents to 1 genomic equivalent, where 1 genomic equivalent corresponds to the total amount of DNA in a single normal human male diploid cell (6.4pg). The upper panel shows the MDA reactions. The lower panel shows the PCR reactions. Prior to PCR, MDA products were diluted to a total DNA concentration of 0.025GEq original input material. (B) Agarose gel analysis of MDA and PCR reactions a series of 1, 2, 5 and 10 normal human diploid fibroblasts. The upper panel shows the MDA reactions. The lower panel shows the PCR reactions. The MDA products were diluted to 0.025GEq original input material prior to PCR. Uncut λ DNA (48kb) was included as a size marker on the gel.
79 It has generally been accepted that 10 or more cells should be the minimum amount of DNA added to an MDA reaction (Sorensen et al.,
2004; Dean et al., 2002). Although the manufacturer recommends a minimum of 1ng to produce reliable results (Amersham Biosciences,
2003a), results show that as little as 1 human cell can be amplified by
MDA and used in subsequent downstream applications such as single locus PCR. MDA amplification of the single cell samples did not require purification of the cells. The initial denaturation step of 95ºC for 3 minutes that is included in the MDA protocol appeared to be adequate for the lysis of up to 10 cells, and subsequent cellular components did not appear to be inhibitory to the MDA reaction. It was also found that the MDA products did not require purification prior to PCR amplification. The ability to bypass a purification step allows direct use of the MDA products in downstream assays, therefore simplifying sample processing and automated procedures.
The production of high molecular weight DNA was observed in MDA negative control reactions that did not contain DNA template. Non- specific amplification product in MDA negative controls is expected according to the manufacturers (Amersham Biosciences, 2003a), and this product does not perform in downstream applications due to the absence of human DNA sequences (Amersham Biosciences, 2003a;
Amersham Biosciences, 2003b). It was confirmed that no-template
MDA reactions did not generate PCR amplicons and comparative genomic hybridisation (CGH) of this MDA product against normal
80 human metaphases did not result in hybridisation (results are presented in Chapter 4, section 4.2.1). Due to its sensitivity, MDA would readily amplify low levels of contaminant genomic DNA. In order to prevent the occurrence of contamination, it was thus important to perform MDA work under strict environmental conditions based on those routinely used in ancient DNA testing (O’Rourke et al., 2000). Furthermore, as per any other genetic assay, the inclusion of negative (no template) controls is essential to monitor contamination events in MDA reactions.
Negative controls were included in every round of MDA during the investigation and were found to be free of human DNA sequences in all instances, as determined by PCR or whole genome CGH hybridisation.
Table53.1. Amplification fold and final yield for single cell samples amplified by MDA.
No. of Amplification Total Yield* Recommended Final Cells Fold Dilution Yield* 1 100 2,000 1:100 2,000 2 10,000 200,000 1:1,000 20,000 5 10,000 200,000 1:1,000 20,000 10 10,000 200,000 1:1,000 20,000 Yield† 5µg *The yield is expressed in the approximate number of single locus PCR reactions (1µL of DNA template in a 25µL PCR reaction) that can be performed using diluted product from a 20µL MDA reaction. The yield is related to the inverse of the end-point dilution, as determined by serial dilution of the MDA product followed by PCR amplification of all dilutions. †Typical yield as determined by spectrophotometric quantitation.
It was observed that a greater amount of PCR amplicon was generated from the lower DNA concentrations as assessed by gel electrophoresis
81 (Figure 3.1). The typical DNA yield from MDA reactions as determined by spectrophotometric quantitation did not vary between different starting amounts of template, with consistent yields of about 5µg for every 20µL MDA reaction (Table 3.1). This finding is consistent with prior observations of yields between 20-30µg for 100µL MDA reactions
(Dean et al., 2002). The yield consistency of MDA reactions, despite variability in the quantity of the original input DNA, provides increased reproducibility in downstream applications as it minimises the need for extensive normalisation to adjust DNA concentrations prior to genetic analysis. For forensic DNA applications, this is especially advantageous as it reduces sample handling time and minimises the occurrence of cross-contamination by reducing sample handling, pipetting, tube transfers, etc and thus maintaining sample integrity.
Figure53.2. Agarose gel analysis of product from the PCR amplification of DNA template from a two-cell MDA reaction diluted by 10-fold serial dilution to 1:1,000,000.
82 The MDA product of a single human fibroblast was diluted down to
1:100 and resulted in successful PCR amplification. For a 20µL MDA reaction, the amount of DNA generated by MDA of a single cell yields sufficient DNA template for up to 2,000 individual PCR reactions (Table
3.1). The MDA of two single human fibroblasts was found to amplify single locus targets by PCR even after diluting to 1:1,000,000, theoretically generating sufficient template for up to 20,000,000 PCR reactions (Figure 3.2). However, PCR amplification of MDA products from two or more cells was more reproducible when the MDA products were diluted to 10-3, for a conservative total yield of 20,000 PCR reactions from each sample (Table 3.1).
MDA results in a higher yield in terms of the total number of downstream PCR reactions possible, in contrast to that of DOP-PCR
(400-500 PCR reactions from 10ng of input DNA) (Bannai et al., 2004;
Barbaux et al., 2001; Cheung and Nelson, 1996) and PEP (100 PCR reactions from 5ng of input DNA) (Bannai et al., 2004). However, due to differences in PCR assay conditions throughout the literature on
WGA yields, a direct comparison is difficult to make. Nevertheless, a large number of PCR reactions are possible from as little as two individual cells after MDA, which enables repeated rounds of retesting, independent confirmatory testing and sample archiving. It also allows the sample to be subjected to different downstream applications in order to increase the amount of information obtainable from that sample. This is in contrast to subjecting a limited sample, in the order of one or two
83 single cells, to a particular assay that can only be performed once due to sample availability. In this case, reaction failure prevents any chance of obtaining data from that sample. The large amount of DNA template produced by MDA is also advantageous as it provides sample consistency, because a variety of assays can be performed on one single sample source, without the need to re-extract a sample for each application.
3.2.2. MDA of Simulated Forensic Samples
The investigation on the utility of MDA in forensic biology proceeded with a range of simulated forensic samples that included fresh buccal swabs, finger swabs, swabs from touched pens, licked stamps, used cigarette butts and single shed hairs. All samples were obtained from laboratory colleagues with informed consent and within the guidelines and approval of the Queensland University of Technology Human
Resources Ethics Committee.
A decision on which extraction chemistry to perform on the forensic samples was made early in the project, based on observations made from the results of amplifying single cell samples as described in section
3.2.1. Because MDA amplification did not require purification of the single cell samples, and was sensitive down to the single genome level, it was hypothesised that simulated forensic samples would only need minimum preparation prior to MDA. Furthermore, as long as the sample
84 contained at least 1 genome equivalent of amplifiable DNA, it was hypothesised that MDA would be successful. Therefore, it was decided that only simplified extraction protocols were required. However, in order to achieve maximum success, the simplified extraction protocols were required to satisfy the following criteria: (1) maximum DNA yield;
(2) ability to remove inhibitors; (3) fast and simple with minimum handling; and (4) economically viable. Furthermore, the use of a widely accepted extraction procedure, specifically one that is routinely used and validated for forensic applications, was considered to be advantageous, as it would reflect a real-world situation. Based on discussions with laboratory colleagues and staff from the John Tonge Centre for Forensic
Biology, Queensland Health Scientific Services (Coopers Plains,
Brisbane, QLD Australia), it appeared that the Chelex-100 extraction procedure (Walsh et al., 1991) is widely accepted in the forensic community. Thus, modifications of the 5% Chelex-100 protocol (hereby referenced to as 5% Chelex) were carried out.
Because the GenomiPhi™ MDA system requires 1µL of input DNA, the ability of the extraction procedure to consistently yield a sufficient concentration of DNA was considered important, as further concentration of the sample, for example by Microcon membrane concentration (Millipore Corp., Billerica, MA, USA), would not be required. Furthermore, additional handling of the sample also increases the chances of contamination.
85 Initially, single-tube extractions were performed in 200µL volumes of
5% Chelex but the resulting concentration of DNA sample was too dilute, as determined by amplifying 1µL of the sample by single locus
PCR and agarose gel electrophoresis (data not shown). Reducing the volume to 50µL resulted in higher concentration of the DNA sample and better performance in PCR amplification, based on agarose gel results
(data not shown). Modification of a low-volume buffer digest protocol, based on a procedure published previously (Allen et al., 1998), was also performed. It was found that the modified extraction methods performed satisfactorily throughout the analysis. It was identified that each extraction procedure was more suitable for certain sample types, and some sample types could be extracted using different protocols.
Observations made during the assessment are presented in Table 3.2.
86 Table63.2. Assessment of simplified extraction protocols for use with forensic samples. Procedure Protocol* Protocol Properties Sample Types† 5% Chelex (200µL) 1. Add 400µL of ET buffer to sample in a 0.5mL tube. Suitable for larger Blood stains, bloodspots 2. Incubate at room temperature for 30 minutes. Vortex for 5 samples containing higher on paper (FTA), whole seconds. concentration of cotton swabs. 3. Spin on tabletop centrifuge at 13,000rpm for 1 minute. inhibitors; final DNA 4. Taking care not to disturb the pellet, remove and discard sample is quite dilute; sufficient supernatant to leave the pellet submerged. Leave the protocol complete in sample matrix with the pellet. ~1hour 15 minutes. 5. Using a widebore pipette tip, add 200µL of 5% (w/v) Chelex suspension (ensure Chelex is evenly distributed). 6. Incubate at 56°C for 30 minutes. Vortex for 10 seconds. 7. Incubate at 99°C for 8 minutes. Vortex for 10 seconds. 8. Spin on tabletop centrifuge at 13,000rpm for 3 minutes.
5% Chelex (50µL) 1. Using a widebore pipette tip, add 50µL of 5% (w/v) Chelex Suitable for small samples Touched objects suspension to sample in a 0.5mL tube. with low concentration of (swabbed), finger and 2. Incubate at 56°C for 30 minutes. Vortex for 10 seconds. inhibitors; final DNA buccal swabs, cigarette 3. Incubate at 99°C for 8 minutes. Vortex for 10 seconds. concentration is high; butts, licked stamps and 4. Spin on tabletop centrifuge at 13,000rpm for 3 minutes. quick procedure (~50 envelopes. minutes). Buffer digest (20µL) 1. Add 20µL of 1x Platinum® Taq PCR buffer to sample in tube. Suitable for small samples Shed hairs (4cm 2. Add 2µL of Proteinase K (20mg/mL). with low concentration of lengths), cigarette butts, 3. Centrifuge briefly and incubate at 56°C for 3 hours. inhibitors; less hands-on licked stamps and 4. Inactivate Proteinase K at 95°C for 10 minutes. time; final DNA envelopes. 5. Spin on tabletop centrifuge at 13,000rpm for 30 seconds. concentration is high; protocol complete in ~3 hours 15 minutes. * For detailed protocols, please refer to Chapter II: Materials and Methods. † Some extracted samples required dilution prior to MDA.
87 As a quality control measure, MDA products (diluted 1:2) were visualised on a 1.5% agarose gel to ensure that high molecular weight
DNA (present as a visible smear) was generated in the MDA reaction.
The presence of amplifiable DNA was confirmed by diluting the MDA product at 1:200 in ET buffer (0.1mM EDTA, 0.1mM Tris-HCl, pH 7.5) and amplification by PCR. If the expected PCR product was present, the original MDA product was serially diluted 10-fold down to 1:1,000,000 in ET buffer and amplified by PCR to determine a dilution end-point.
Both end-point PCR and quantitative real-time PCR were used.
After MDA, simulated forensic samples were amplified by at least 200- fold and up to 200,000-fold (Table 3.3). MDA products of forensic samples could be diluted down to 1:100,000, thus providing sufficient template for up to two million PCR assays for the detection of single copy genomic and mitochondrial DNA targets, from MDA of only 1µL extracted DNA (Figure 3.3).
Table73.3. Amplification fold and maximum yield for various types of simulated forensic specimens from MDA of 1µL extracted DNA. Sample Type Amplification Fold Maximum Yield* Touched pen (n=9) 200 – 200,000 2,000,000 Shed hair (n=5) 200 – 20,000 200,000 Licked stamp (n=9) 200 – 2,000 200,000 Finger swab (n=10) 200 – 2,000 200,000 Buccal swab (n=2) >200 >4,000 *The yield is expressed in the approximate number of PCR reactions (1µL of DNA template in a 25µL PCR reaction) that can be performed using diluted product from a 20µL MDA reaction. The yield is related to the inverse of the end-point dilution, as determined by serial dilution of the MDA product followed by PCR amplification of all dilutions.
88
Figure63.3. Amplification fold and yield after MDA of simulated forensic samples. The X-axis represents the sample type and the Y-axis represents the fold increase in amplification.
Figure73.4. Comparison of successful single-locus PCR of swab samples, with and without MDA. PCR was performed on the original 5% Chelex extract (“CX”) of a fresh swab sample and the GenomiPhi™ MDA product from the same sample (“GP”). Top panel includes the results from agarose gel electrophoresis after amplification of a 194bp human mitochondrial target (mtDNAg16391a), and the bottom panel are results from amplifying a 102bp human nuclear DNA target (TYRP1). Samples include: “SW A” dry thumb and index fingers (donor S); “SW B” pen swab (donor S); “SW C” dry thumb and index fingers (donor J); “SW D” ENTER and SPACEBAR keys of a computer keyboard (donor A), “SW E” metal door handle; “SW +” buccal swab positive control (donor R); “SW -” sterile swab negative control (no DNA). Sub-samples of both the Chelex extracts and MDA products were diluted proportionately to 1:50 of original input DNA material prior to PCR amplification.
89 Figure 3.4 illustrates successful PCR amplification of MDA-amplified swab samples, compared to their original un-amplified Chelex extracts.
Data suggest that successful downstream PCR amplification of MDA products was the result of MDA-generated DNA material, and is not attributed to the original input DNA that was added to the MDA reaction.
Subjecting 20µL of DNA extract to MDA can theoretically yield sufficient template DNA for a large number of tests, in excess of
4,000,000 PCR reactions. Although one might not require such large amounts of DNA template, MDA effectively provides an unlimited supply of template DNA for downstream analysis of low copy number samples, including a variety of forensic samples.
Inhibition was not encountered in the MDA reaction during amplification of single cells, purified human genomic DNA or simulated forensic samples. Slight inhibition was observed in MDA reactions containing licked stamp extracts, in the form of reduced yield in high molecular weight product as determined by gel electrophoresis, possibly due to the adhesive component (data not shown). However, inhibitors in a DNA extract were rendered ineffective by dilution prior to MDA.
Since MDA was consistently successful for samples as little as 1 to 2 cells, sample dilution was not a concern.
90 3.2.3. The Effect of Degraded DNA Templates on GenomiPhi™ MDA
High molecular weight genomic DNA was degraded in reactions
containing bovine pancreatic DNase I to produce a fragmentation
gradient corresponding to the input concentration of DNase I enzyme.
DNA samples were most fragmented in reaction 1, containing the
highest DNase concentration of 0.1 units (U), and least fragmented in
reaction 5, with the lowest DNase concentration of 0.00625U. The
degraded fragments varied in length from less than 100bp to greater than
3000bp (Table 3.4). Fragment sizes generated by the artificial
degradation reaction were confirmed via PCR amplification of different
amplicon lengths (Figure 3.5), using the following primers: genomic
DNA primers TYR (497bp), CITED1 (380bp), OA1 (233bp), TYRP1
(102bp), and mtDNA primers g16391a (194bp) and g1719a (112bp).
Table83.4. Fragment size ranges of degraded DNA, artificially fragmented by a dilution of bovine pancreatic DNase I enzyme. Reaction [DNase] Fragment nuDNA amplicons mtDNA number sizes (bp) amplicons TYR CITED1 OA1 TYRP1 g16391a g1719a 497bp 380bp 233bp 102bp 194bp 112bp 1 0.1U No smear 2 0.05U ~100-200 3 0.025U ~200-500 4 0.0125U ~500-3,000 5 0.00625U >3,000
91
Figure83.5. Agarose gel analysis of artificially-degraded genomic DNA. Left hand panel: high molecular weight genomic DNA was fragmented by a two- fold dilution series of bovine pancreatic DNase I (reaction 1: 0.1U enzyme; 2: 0.05U; 3: 0.025U; 4: 0.0125U; 5: 0.00625U). Right hand panel: after purification of artificially-degraded DNA, fragment sizes were confirmed by PCR amplification using primer sets that generate amplicons of different sizes, ranging from 102bp to 497bp.
As indicated in Table 3.4 and Figure 3.5, PCR yielded products for most samples except in reactions 1 and 2 where the DNA appeared to be too fragmented to allow amplification of the larger (greater than 233bp)
OA1, CITED1 and TYR regions. All degraded DNA samples were successfully amplified using the smaller TYRP1 and mtDNAg1719a primers.
92
Figure93.6. Artificially degraded DNA fragments after amplification by MDA. Samples 1 to 5 contained DNA fragmented by a dilution of bovine pancreatic DNase I (1: 0.1U enzyme; 2: 0.05U; 3: 0.025U; 4: 0.0125U; 5: 0.00625U). Samples P and N are positive and negative controls respectively. Uncut λ DNA (48kb) was included as a size marker on the gel to illustrate the high molecular weight DNA product generated by the MDA amplification of fragmented templates.
MDA amplification was successful for all degraded samples (Figure
3.6). Following MDA, diluted MDA products were subjected to PCR using TYRP1 and mtDNAg1719a primer sets, with amplicons around
100bp in size (Figure 3.7). PCR was successful for all MDA products except those from reactions 1 and 2 that contained fragments less than
200bp (Table 3.5). PCR of the mitochondrial target was more successful than nuclear DNA for reaction 2, probably due to the larger number of mitochondrial genomes (Bogenhagen and Clayton, 1974). PCR amplicons were generated from MDA products diluted to 1:1,000 and
1:10,000, generating a total yield of at least 20,000 single locus PCR reactions from DNA fragments as short as 200-500bp in one single
MDA reaction.
93
Figure103.7. PCR analysis of the MDA reactions shown in Figure 3.6. PCR analysis was conducted using the 102bp TYRP1 nuclear DNA fragment and the 112bp g1719a mitochondrial DNA marker.
Table93.5. Results for PCR of artificially degraded fragments pre- and post-MDA using short (approximately 100bp) PCR primer sets.
Reaction Fragment sizes Pre-MDA Post-MDA number (bp) TYRP1 g1719a TYRP1 g1719a 102bp 112bp 102bp 112bp 1 No smear 2 ~100-200 3 ~200-500 4 ~500-3,000 5 >3,000
These results suggest the possibility of using MDA for the amplification of moderately fragmented DNA templates for applications that require large amounts of sample such as SNP analysis, or sample archiving of precious DNA samples that may be degraded, for example the storage of archaeologically-significant DNA specimens. However, the results also show that MDA will not successfully amplify highly degraded fragments less than approximately 200bp in size. This is presumably because the
94 degraded DNA fragments (less than 200bp) are too short to allow multiple random hexamers to bind and maintain MDA reaction kinetics.
Furthermore, if primers bind close to the middle of the fragments and are extended by φ29 DNA polymerase, each successive strand displacement event would generate shorter templates until primer binding and extension cannot proceed. The lack of amplifiable DNA template would therefore promote the formation of primer-primer duplexes and primer concatemers.
In cases where forensic DNA samples are highly fragmented, the specimens should be utilised in an assay where the maximum amount of information is obtainable, for example an STR kit that amplifies and detects degraded DNA fragments, such as the BodePlex miniSTR systems that were used to identify World Trade Center victims (Holland et al., 2003; Schumm et al., 2004). The BodePlex 1 and BodePlex 2 miniSTR systems utilise primers from the larger AmpFlSTR® Profiler
Plus® and COfiler® systems that were re-designed into smaller amplicons with optimized performance for degraded DNA templates and reduced template concentrations of 0.25ng (Schumm et al., 2004). The use of miniplexes is preferable in the case of degraded DNA samples, compared to using conventional multiplex PCR reactions that may generate partial genetic profiles, locus drop-out and loss of signal
(Whitaker et al., 1995), or mtDNA testing that produces less powerful data compared to STR multiplexes, due to the non-Mendelian nature of mtDNA inheritance (Butler and Levin, 1998).
95
The utility of MDA products from limiting forensic samples in downstream short tandem repeat (STR) multiplex PCR, as routinely used in forensic biology for human identification purposes, should be considered for a future study. Furthermore, the future study should incorporate an analysis into the conservation of STR allele ratios in mixed DNA samples, pre- and post-MDA, to determine the presence of any amplification bias within the MDA system.
3.2.4. Archiving of precious population samples
The utility of MDA-based WGA products as archival DNA storage samples has not previously been investigated. Theoretically, the large amount of DNA generated by MDA would provide a sufficient amount of sample for a wide range of genetic analyses or future testing.
Although room-temperature DNA storage media such as FTA® cards
(Whatman plc, Kent, UK) and IsoCode® Stix (Schleicher and Schuell,
Dassel, Germany), or non-cryogenic preservation of tissues in dimethyl sulfoxide (DMSO) and ethanol (Kilpatrick, 2002), are routinely used for archiving DNA samples, MDA products stored at -20°C or -70°C may persist for a longer period of time in a more satisfactory state as the
DNA templates are isolated from environmental factors or harsh chemicals that may potentially damage the DNA sample. Complex repositories of whole blood specimens (present as bloodspots or biological “dog tags”), cryopreserved tissue and protein samples, buccal
96 epithelial cells and Epstein-Barr virus transformed lymphocytes may be made more efficient by the utility of MDA products as DNA archival material. Biological specimen banks for epidemiological studies may also gain from MDA, due to the requirement for good DNA quality and quantity, ease of collection, storage and the ability to accommodate future needs for genotyping (reviewed in Steinberg et al., 2002).
This section outlines the results of Aim I. Precious population samples consisted of single bloodspots on various types of matrices, obtained from members of ethnic populations including American Caucasian,
African American, Apache Indian, American Hispanic, Spanish Basque, and Indigenous Australian. Spanish Basque bloodspots were a gift from
Dr José A. Lorente (Department of Legal Medicine, University of
Granada, Spain), and Indigenous Australian samples were donated by
Assoc. Prof. Leo Freney (Forensic Biology, Queensland Health
Scientific Services, Brisbane QLD, Australia). The remaining population samples were a gift from Dr Tamyra R. Moretti of the Federal Bureau of
Investigation (FBI, Quantico VA, USA). Information on the actual age of individual bloodspots samples was not available at the time, but all samples were at least 4 years old at the time of this investigation. All bloodspots were stored dry in either plastic Ziploc bags or paper envelopes, at room temperature in the dark.
Bloodspots were extracted using the 5% Chelex (200µL) protocol as outlined in Table 3.2. This method was preferable over the low-volume
97 5% Chelex (50µL) alternative, as it allowed better removal of inhibitors
such as heme. PCR amplification of DNA extracted from FBI population
samples was unsuccessful for fragments greater than 100bp, suggesting
possible degradation of the DNA in the sample (Table 3.6). In contrast,
PCR amplification of Spanish Basque and Indigenous Australian
samples successfully generated uniform amplification of 100bp
(Agouti), 380bp (CITED1) and 497bp (TYR) fragments (Table 3.6).
Table103.6. PCR amplification of extracted ethnic population samples.
Sample ID Ethnicity Matrix 497bp* 380bp* 100bp* C16 Caucasian Cotton Cloth C27 Caucasian Cotton Cloth C53 Caucasian Cotton Cloth C77 Caucasian Cotton Cloth AA629 Afr. Am. Cotton Cloth AA630 Afr. Am. Cotton Cloth AA680 Afr. Am. Cotton Cloth AA682 Afr. Am. Cotton Cloth H321 Hispanic Cotton Cloth H322 Hispanic Cotton Cloth H326 Hispanic Cotton Cloth H350 Hispanic Cotton Cloth A001 Apache Cotton Cloth A002 Apache Cotton Cloth A076 Apache Cotton Cloth A097 Apache Cotton Cloth AB22 Indig. Austr. Cotton Cloth-like AB39 Indig. Austr. Cotton Cloth-like AB66 Indig. Austr. Guthrie-like AB74 Indig. Austr. Guthrie-like SB23 Sp. Basque Thin Paper Card SB32 Sp. Basque Thin Paper Card SB43 Sp. Basque Thin Paper Card SB47 Sp. Basque Thin Paper Card *Number of ticks () indicates intensity of PCR product on a 0.5X TAE 1.5% agarose gel, stained by ethidium bromide and viewed by UV illumination: () indicates a band is present but faint; () indicates band is present and highly visible; () indicates a band of very strong intensity is present. Crosses () represent no visible PCR product.
98 Extraction of the degraded FBI populations samples was attempted using other extraction or pre-treatment options such as methanol/acetone pre- treatment (Hong et al., 1999), alkaline lysis (Klintschar and Neuhuber,
2000), and alkaline lysis with direct MDA (Amersham Biosciences,
2003a). Other extraction procedures attempted were those that are routinely applied to degraded forensic or archaeological sources such as guanidinium thiocyanate (Hlinka, unpublished PhD thesis) and an in- house extraction buffer containing high concentrations of ethylenediaminetetraacetic acid (EDTA), sodium dodecyl sulphate
(SDS) and three non-ionic detergents (Dr Tom Loy, pers.comm.).
However, these alternative extraction methods did not improve the quality of extracted DNA from the FBI bloodspot samples (data not shown).
Due to sample degradation, MDA of the FBI samples could not be performed. However, Spanish Basque and Indigenous Australian samples could be amplified by MDA and yielded template DNA suitable for use in downstream PCR amplification. This observation supports the previous finding that MDA requires a minimum DNA template length of at least 200bp to ensure successful amplification, as described in Section
3.2.3.
To test the utility of MDA as an archiving tool for bloodspot samples, a set of fresh bloodspots was prepared. A small database of bloodspots from Asian donors was created on Guthrie paper and used in the analysis
99 within 1 month of collection. Asian bloodspot samples were extracted using the 5% Chelex (200µL) protocol (Table 3.2) and success of the extraction procedure was confirmed by PCR amplification (data not shown). However, MDA of the fresh Asian bloodspot extracts was unsuccessful as indicated by the presence of very faint smears on agarose gels and unsuccessful downstream PCR amplifications. It appeared that the MDA reactions failed to yield product, possibly due to inhibition by an excess of heme in the fresh bloodspots that was not efficiently removed during the Chelex extraction procedure. However, the effect of inhibition was generally overcome by diluting the extract in
ET buffer (0.1mM EDTA, 0.1mM Tris-HCl, pH 7.5), and adding 1µL of this diluted sample to MDA (Figure 3.8).
Downstream PCR amplification was most successful when the fresh bloodspot extract was diluted 1:10 prior to MDA, and was able to generate amplicons down to 1:200 of diluted MDA product (Figure 3.9).
Based on these data, subjecting 1µL bloodspot extract (diluted 1:10) to a
20µL MDA reaction potentially yields sufficient DNA template for at least 4,000 single locus PCR reactions (Table 3.7). A scalable MDA reaction containing 10µL of bloodspot extract (diluted 1:10) would allow at least 40,000 single locus PCR reactions.
100
Figure113.8. Inhibition of bloodspot samples. MDA of fresh Asian (Australian) bloodspots exhibited inhibition when 1µL of a neat (1:1) dilution was added to the MDA reaction, as indicated by a faint smear, suggesting low product yield. Diluting a sub-sample of the extract in ET buffer (0.1mM EDTA, 0.1mM Tris-HCl, pH 7.5) appeared to reduce the effect of the inhibitors in the MDA reaction. Uncut λ DNA (48kb) was included as a size marker on the gel.
Figure123.9. Downstream PCR amplification of MDA reaction products from diluted bloodspot extracts. Diluting a fresh bloodspot extract by 1:10 prior to MDA generated DNA template that could be amplified in downstream PCR at 1:100 and 1:200 dilutions of the MDA product.
101
Table113.7. Potential total yield from MDA of diluted fresh bloodspot extracts (in number of single locus PCR reactions). Bloodspot sample dilution and No. of single locus PCR No. of single locus PCR volume reactions before MDA reactions after MDA 1:10 (1µL) 1 4,000 1:10 (10µL; scalable MDA) 10 40,000 50uL extract, diluted 1:10 500 2,000,000 (500µL total; scalable MDA)
The success of MDA and the high yield of synthesised product suggest
that MDA is a suitable tool for the archiving of precious and limited
DNA samples such as Chelex-extracted single bloodspots. MDA was
able to generate large amounts of template DNA that is suitable for
downstream use in applications such as single locus PCR. However, as
outlined in Section 3.2.3, the success of MDA as an archiving tool is
dependent on the quality of the original starting material. MDA cannot
be performed if the sample is significantly degraded or if it contains an
excess of inhibitors. The former issue cannot be remedied without the
aid of a DNA repair system, which is beyond the scope of this thesis, but
the latter can be overcome by simply diluting the sample by 1:10 in ET
buffer prior to use in MDA.
3.3. Summary and Conclusions
As outlined in this chapter, MDA is a DNA amplification technique that
is sensitive to very low amounts of DNA, down to the single genome
102 level. It was found that MDA was successful as long as a minimum of one single cell, or 6.4pg of genomic DNA, was added to the MDA reaction. MDA consistently generated a final DNA yield of approximately 5µg for every 20µL reaction, confirming observations as published in the literature. MDA products did not require further purification or manipulation prior to downstream applications such as
PCR. PCR products were observed for diluted MDA products of single cell samples, even at dilutions as low as 1:1,000,000. At these dilutions,
MDA can potentially provide sufficient DNA material for up to
20,000,000 diagnostic single locus PCR assays. However, more reproducible yields were obtained from MDA products diluted to
1:1,000 resulting in sufficient DNA product for 20,000 PCR assays. It is evident that a large number of PCR reactions are possible from as little as one cell, enabling many repeated rounds of retesting that was previously limited due to the small sample size. The large amount of
DNA template produced by MDA also provides sample consistency, as a variety of test assays can be performed on one single sample source.
The data outlined in this chapter also suggest that MDA using the
GenomiPhi™ DNA Amplification Kit is a useful tool for the amplification of a variety of forensic samples. MDA was capable of amplifying limiting forensic samples such as DNA on cigarette butts, licked stamps, touched objects, single shed hairs, finger swabs and buccal swabs, extracted using simplified Chelex or buffer digest extraction protocols. It was evident that the simplified extraction
103 protocols were able to liberate sufficient DNA of adequate quality and quantity to enable use in PCR and MDA. MDA was capable of amplifying forensic samples by up to 200,000-fold compared to the original unamplified DNA sample, and generated sufficient DNA for up to 2,000,000 single locus PCR reactions.
A thorough investigation on the use of MDA products in short tandem repeat (STR) multiplex PCR assays, as performed in the discipline of forensic biology for the purposes of human identification, should be considered for a future study. The STR typing system should consist of one that is widely accepted in the field of forensic biology and contains
STR loci that are routinely used for human identification, such as those that make up the Combined DNA Index System (CODIS) as endorsed by the Federal Bureau of Investigation (FBI), and are available in commercial kits such as the AmpFlSTR® Profiler Plus® multiplex STR kit (Applied Biosystems, Foster City, CA, USA). Based on the average
MDA yields identified in this study, one GenomiPhi™ MDA reaction is able to generate sufficient DNA material for at least 5,000 Profiler Plus®
PCR assays containing 1ng of input DNA. Furthermore, an assessment of environmental effects on the samples, such as variable temperature and exposure to the elements as experienced by real forensic samples, and the subsequent effects on MDA success rates, should also be performed. Any future investigations should also include an analysis on the conservation of STR allele ratios in mixed DNA samples, comparing pre- and post-MDA, in order to determine faithful amplification of the
104 mixed samples, and assess the presence of any amplification bias within the MDA system that may alter the allele ratios. This latter investigation is particularly important in the case of mixed DNA samples, such as those encountered in sexual assault samples (fingernail scrapings, vaginal swabs), as the presence and conservation of major and minor
STR profiles may aid forensic investigations. For successful amplification of mixtures from limited forensic samples, MDA must faithfully represent each component of the mixture without any bias.
The success of MDA was dependent on the quality of the original template material. The results outlined in this chapter identified the requirement for DNA templates greater than 200bp in length for use in
GenomiPhi™ MDA. It is presumed that the use of degraded DNA fragments less than 200bp will affect MDA reaction kinetics, as primer binding sites become limited and each successive strand displacement event has the potential to generate shorter templates until primer binding and extension cannot proceed any further. It is therefore essential that in order to ensure successful MDA of precious or limited samples, DNA extracts should be screened for fragments greater than 200 bp. The screening process can be easily performed using a PCR assay for the amplification of at least one single-copy region in the genome of interest. Although this screening process introduces an extra step post- extraction and pre-MDA, it should be considered especially when very limiting or precious forensic evidentiary samples are going to be subjected to MDA.
105
Screening a sample for DNA templates of adequate quality can increase the efficient use of that sample, for example if the forensic sample shows evidence of degradation then it may be used in a miniSTR multiplex system designed for fragmented samples, instead of MDA and conventional STR analysis. In contrast, a sample that passes the screening process can be amplified by MDA, the product of which can be used in a variety of downstream applications that may increase the amount of information obtainable from that sample. Furthermore, the coupling of the MDA protocol with a DNA repair reaction for the amplification of limited and degraded samples should be investigated in a future study. The combination of MDA with a DNA repair mechanism would be especially advantageous for the utility of MDA in the field of molecular archaeology and ancient DNA analysis.
As a potential tool for DNA sample archiving, MDA was able to amplify single bloodspots extracted by the 5% Chelex protocol. However, heme or other blood components appeared to inhibit MDA, as indicated by reduced product yields determined using agarose gel electrophoresis.
Inhibition was generally overcome by diluting the blood extract by 1:10 in ET buffer (0.1mM EDTA, 0.1mM Tris-HCl, pH 7.5) prior to MDA.
MDA of 1µL diluted bloodspot extract produced sufficient DNA template for up to 4,000 PCR reactions, with more DNA able to be produced using scalable MDA reactions. The inability to amplify FBI bloodspot extracts consisting of fragmented DNA further supports the
106 previous observation that MDA requires DNA templates of an adequate quality. In summary, MDA is suitable for applications in sample archiving to generate DNA for confirmatory testing, storage and future work, as long as the original template DNA is non-degraded (greater than 200bp in length) and is of a sufficient quantity (greater than 1 single genome copy per microliter).
Based on observations and results presented in this section, guidelines can be offered to ensure successful utility of MDA, specifically for amplifying limited forensic samples and for application as an archiving tool for limited or precious bloodspot samples.
3.3.1. Guidelines for amplifying limited forensic DNA samples by MDA using the GenomiPhi™ DNA Amplification Kit (Amersham Biosciences,
Piscataway, NJ, USA)
1. Extract forensic exhibit using a suitable extraction protocol for low
copy number DNA samples (for example, the simplified 5% Chelex
and/or buffer digest protocols as outlined in Table 3.2).
2. Screen the DNA extract for fragments greater than 200bp in length,
for example via PCR assays that amplify a 100bp amplicon and a
400bp amplicon.
a. The presence of both (100bp and 400bp) amplicons suggests the
presence of DNA template of suitable quality for MDA.
107 b. The presence of the 100bp amplicon and the absence of the
400bp suggest possible degradation of the DNA sample to
fragments less than 400bp. In this case, the sample should be
used in assays that will maximise the amount of information
obtainable (for example, a miniSTR assay for degraded forensic
samples).
c. The absence of both amplicons can suggest extreme
degradation of the sample, or the presence of inhibitors in the
extract. Dilutions of the extract should be made and the sample
re-screened. If the assay still fails, then that sample is not
suitable for analysis. If a sample passes at a particular dilution,
that dilution of the sample should be subjected to MDA.
d. The screening assay should utilise primers designed for single-
copy regions, specific for the genome under analysis (for
example, human), to ensure appropriate sensitivity and
specificity. The screening assay can be performed using single-
plex PCR (two different PCR reactions) with fragment
detection on agarose gels, or via a multiplex PCR system using
fluorescent primers or via a TaqMan assay utilising fluorescent
probes with fluorescent detection of the amplified products. The
screening assay can incorporate a quantitation system.
3. Samples that pass the screening assay can be subjected to MDA, as
per the manufacturer’s instructions. Positive (known amount of
DNA) and negative (no template DNA) controls should be included.
108
4. Dilute a sub-sample of the MDA products by 1:2 and analyse by
agarose gel electrophoresis (1.5% agarose gel, in 0.5x Tris-acetate-
EDTA agarose gel for 30 minutes at 100V), followed by appropriate
staining and visualisation, to observe the presence of high molecular
weight MDA product. A high molecular weight molecular marker
(for example, uncut lambda DNA) should be included on the gel to
aide visualisation. The absence of high molecular weight MDA
product for a sample may indicate inhibition or MDA reaction
failure. To eliminate the possibility of reaction failure, assess the
yield of the MDA positive and negative controls (both controls
should yield visible product).
5. Dilute a sub-sample of the original MDA product by 1:200 in ET
buffer (0.1mM EDTA, 0.1mM Tris-HCl, pH 7.5) and perform
single-locus PCR to assess successful MDA of the genome of
interest. Upon successful PCR amplification, the MDA product can
be further diluted.
6. As an alternative to Guideline 5, the 1:200 MDA dilution can be
subjected to a quantitative real-time PCR assay, such as the
Quantifiler™ Human DNA Quantitation kit (Applied Biosystems,
Foster City, CA, USA), if the MDA product is to be used for
downstream STR assays. Quantitation is required prior to use in
multiplex STR PCR, as the PCR conditions are optimised for a
109 certain amount of input DNA. For example, 1-2.5ng of DNA is
required in a 50uL PCR reaction using the AmpFlSTR® Profiler
Plus® multiplex STR kit (Applied Biosystems, Foster City, CA,
USA). Accurate quantitation prevents overload or excessive
reworking of the STR PCR reaction.
7. For non-PCR-based quantitation procedures, such as by
spectrophotometric absorption (A260/A280), the MDA product must
be purified prior to quantitation in order to remove protein
components and residual primers. Purification of the MDA product
is also recommended for enzyme-based quantitative assays, such as
the AluQuant® system (Promega Corp., Madison, WI, USA).
Amersham Biosciences claim that quantitation by the intercalating
agent PicoGreen® (Molecular Probes, Eugene, OR, USA) does not
require purified MDA product.
8. MDA product should be stored at 4°C or -20°C for short-term or
long-term storage respectively. Working dilutions (for example,
1:200) can be stored at 4°C.
9. When using frozen MDA product, the sample should be completely
thawed at room temperature, gently mixed by vortexing to ensure
uniform concentration of MDA product and then centrifuged
briefly.
110 3.3.2. Guidelines for the utility of MDA for archiving fresh bloodspot samples, using the GenomiPhi™DNA Amplification kit (Amersham
Biosciences, Piscataway, NJ, USA)
1. Extract a 5mm x 5mm (or 9mm punch) portion of bloodspot by 5%
Chelex, briefly:
i. Incubate in 200µL ET buffer (0.1mM Tris, 0.1mM EDTA, pH
7.5) at room temperature for 30 minutes.
ii. Vortex, then centrifuge at maximum rpm for 1 minute.
iii. Remove all but 20µL of supernatant. Do not disturb the pellet
and bloodspot matrix.
iv. Add 200µL of uniformly-suspended 5% Chelex and incubate at
56°C for 30 minutes. Vortex briefly.
v. Incubate at 99°C for 8 minutes. Centrifuge at maximum rpm for
3 minutes.
2. For fresh bloodspots, dilute DNA extract by 1:10 in ET buffer for
use in MDA. For older bloodspots, screening the sample (as per
Guideline 2, in Section 3.3.1) may be required.
3. Use 1µL of diluted DNA extract in a 20µL GenomiPhi™ MDA
reaction. Perform MDA as per the manufacturer’s instructions.
4. Examine for the presence of high molecular weight MDA product
on an agarose gel (as per Guideline 4, in Section 3.3.1)
111
5. For archiving, dilute the MDA product to 1:50 with ET buffer and
store at -20°C. Aliquots of MDA product at this dilution can be
made into several tubes and stored in separate freezers for future
use.
6. For single-locus PCR, dilute the MDA product to 1:200 final
dilution using ET buffer and use 1µL sample per PCR reaction.
7. For use in STR multiplex PCR, the sample should be quantified
prior to use as outlined in Section 3.3.1, Guidelines 6-7.
112 CHAPTER FOUR
APPLICATION OF MULTIPLE DISPLACEMENT
AMPLIFICATION IN CLINICAL DIAGNOSTICS
4.1. Introduction
The difficulty in providing a template DNA sample of adequate quality and quantity is encountered not only in disciplines such as forensic biology and molecular archaeology, but also in the field of clinical pathology, especially single-cell molecular diagnostics such as the analysis of isolated single foetal cells from fertilised embryos or DNA from maternal plasma. The 5-10pg of DNA present within a single human cell is often insufficient for extensive downstream testing.
Disease diagnosis of isolated single cells or limited DNA samples has met with little success due to the lack of sufficient DNA template available for use, and therefore a whole genome amplification (WGA) step is often required prior to analysis. The ability to synthesise adequate quantities of highly uniform template DNA from the DNA in single cells would allow extensive genetic analysis by genome screening methods such as comparative genomic hybridisation (CGH) or preimplantation
113 genetic diagnosis (PGD), both of which depend on an ample supply of genetic material of adequate quality.
As described in Chapter 1 (refer to Section 1.2.2.3; see also Figure 1.2),
CGH is a very powerful recent development of fluorescence in situ hybridisation (FISH) (Kallioniemi et al., 1992). CGH can achieve global screening of a DNA sample for the identification of aneuploidy, triploidy and insertions or deletions of chromosomal DNA that is often unresolvable by G-banding techniques (Levy and Hirschhorn, 2002).
CGH is advantageous over other cytogenetic techniques such as FISH and whole chromosomal painting (WCP), as the origin of extra or missing DNA material can be mapped to specific G-bands within the short (p) and long (q) arms of all 23 pairs of chromosomes.
Unlike conventional G-banding, CGH allows direct analysis of genomic
DNA from a sample and thus removes the need to generate metaphase chromosomes from cultured cells (Kallioniemi et al., 1992; Kallioniemi et al., 1994). CGH can detect changes present in as little as 30-50% of specimen cells in a single hybridisation (Kallioniemi et al., 1994), but cannot be used to detect balanced translocations and inversions that do not change the actual copy number of the chromosomal region under analysis (Levy and Hirschhorn, 2002). CGH can be performed either on a glass microscope slide containing immobilised human metaphase chromosomes, or on an array containing complementary DNA (cDNA) sequences or bacterial artificial chromosomes (BAC). The latter
114 potentially increases the assay resolution as particular chromosomes can be interrogated with higher discrimination.
CGH involves generating fluorochrome-labelled probes from a test or patient DNA sample via a nick translation reaction, where nicking of the sample DNA template results in fragmentation of the DNA to between
300-1,000bp and integration of SpectrumGreen™ fluorochrome. The patient DNA is purified and precipitated with an equimolar amount of normal reference DNA labelled with SpectrumRed™ fluorochrome, in the presence of Cot1 DNA to suppress cross-hybridisation between repetitive DNA sequences. Probes are hybridised to normal human metaphase chromosomes on a glass slide, or cDNA arrays if performing array-CGH, after incubation at 37°C for at least 48 hours. The slide is washed to remove unbound probes. Individual metaphase chromosomes are viewed using a fluorescence microscope with an attached CCD camera, aided by visualisation software on a computer. The software combines the total fluorescence from both test (SpectrumGreen™) and reference (SpectrumRed™) DNA probes that are hybridised to the individual chromosomes, displaying the result in a CGH profile. Excess green fluorescence at a particular region indicates an excess or gain of
DNA in the test sample, whereas an excess of red fluorescence indicates loss of chromosomal material. A ratio close to 1:1 indicates normal
DNA dosage (Figure 4.1; also see Figure 1.2). CGH is a time-consuming process that takes up to one week to perform and the success or failure of the test cannot be determined until visualisation of the slide is
115 performed. Failure of a CGH test can arise for a number of reasons.
These may include: non-uniform fragmentation of sample DNA, therefore causing inefficient and insufficient hybridisation of DNA probes; insufficient purification of patient DNA probes, or loss of patient
DNA probes during purification; reagent/chemical failure; and insufficient washing of unbound probes. A CGH test can only be repeated if sufficient DNA sample is available.
Figure134.1. A diagram depicting representations of all 23 pairs of human chromosomes and the CGH profile of a patient DNA sample. In this example, CGH has identified a loss of genetic material in the patient sample towards the telomeric end on the short arm of chromosome 12 (12p), as indicated by the absence of patient (green) fluorescence and an increase of reference (red) fluorescence.
116 CGH is primarily used for the characterisation of genomic imbalances in different tumour and cancer types such as bladder cancer (Kallioniemi et al., 1995), prostate cancer (Visakorpi et al., 1995), and breast cancer
(Tirkkonen et al., 1998). More recently, CGH has become a useful tool for the diagnosis of conditions related to abnormal amounts of cytogenetic material that manifest in the phenotypes described in Table
4.1, including Down’s syndrome (Shaw-Smith et al., 2004), Cri du Chat syndrome (Levy et al., 2002), DiGeorge syndrome (Mantripragada et al.,
2004), Klinefelter syndrome (Geschwind et al., 1998) and Turner syndrome (Christiaens et al., 2000).
Table124.1. Major disorders of the autosomes and sex chromosomes (adapted from Nussbaum et al., 2001).
Syndrome Chromosomal Phenotypic manifestation disorder Down Trisomy 21; Mental retardation; dysmorphic features: Robertsonian short stature, mouth is open showing translocation protruding tongue, wide gap between first (trisomy 21q) and second toes; congenital heart disease; 15-fold increased risk of leukaemia. Trisomy 18 Trisomy 18 Mental retardation; failure to thrive; severe heart malformation; poor survival. Trisomy 13 Trisomy 13 Growth and mental retardation; severe central nervous system malformations; ocular abnormalities (absence of eyes often occurs); cleft lip; polydactyly. Cri du Chat 5p deletion Facial malformations; severe mental retardation; heart defects. DiGeorge 3mb deletion at Craniofacial abnormalities; mental 22q11 retardation; heart defects. Klinefelter XXY (extra X Tall and thin stature; hypogonadism; in males) underdeveloped secondary sexual characteristics; infertility. XYY XYY (extra Y No abnormal phenotype; increased risk of in males) educational or behavioural problems. Turner Only one X Short stature; gonadal dysgenesis; webbed chromosome in neck; renal/cardiovascular anomalies; females average/above average intelligence.
117 CGH analysis of single cell samples is often not possible as a single
CGH assay requires up to 1µg of DNA template (Hawkins et al., 2002;
Wells and Levy, 2003), which is the equivalent of the total amount of genomic DNA present in approximately 150,000 human cells. Although larger specimens such as tumour biopsies can often provide this much sample, the application of CGH for smaller samples containing individual human cells is difficult.
A variety of methods also exist for the screening of embryonic and foetal material for genetic diseases. Amniocentesis and chorionic villus sampling (CVS) are the current standards for prenatal genetic testing to detect genetic abnormalities during the first 10-16 weeks of gestation
(Wells and Delhanty, 2001). Amniocentesis and CVS are both invasive procedures that carry a significant risk to both the mother and the foetus, as the probability of miscarriage following either procedure can be as high as 2% (Wells and Delhanty, 2001). More alarmingly is the association of CVS to an increase in the incidence of congenital malformation (Wells and Delhanty, 2001). Less invasive techniques for prenatal diagnosis are also under development. Of particular interest is the testing of foetal DNA present at extremely low concentrations in the maternal blood stream (Pertl and Bianchi, 1999).
Preimplantation genetic diagnosis (PGD) is another option for genetic testing of embryos and was introduced as an option for avoiding elective abortions following amniocentesis or CVS. Unlike amniocentesis and
118 CVS, PGD involves the testing of embryos that are outside of the mother’s body. The use of PGD aims to diagnose several inherited diseases by genetic analysis of single cells, biopsied from fertilised human embryos following in vitro fertilisation (IVF), in order to select viable embryos for implantation in to the mother’s uterus. This ensures that “at risk” couples have only unaffected embryos replaced in the uterus, avoiding the termination of pregnancy if the embryo is diagnosed as affected by an inherited disorder later during the gestation period.
PGD requires a cycle of IVF to generate large numbers of embryos in culture, and the removal of single blastomeres from these embryos 3 days after fertilisation for genetic analysis. Embryos that are classified as unaffected by genetic abnormalities can then be transferred in to the mother’s uterus. Due to dependency on IVF, PGD is limited by the low efficiency of IVF, as only 20-30% of couples achieve a successful IVF cycle (Wells and Delhanty, 2001).
PGD can be carried out through a two-step investigation of oocytes using the first and second polar bodies (Verlinsky et al., 1997). This technique is laborious, as all oocytes must be tested although a large number will not fertilise (Wells and Delhanty, 2001). Furthermore, polar body analysis detects only maternally inherited mutations and thus the father’s genetic contribution cannot be interrogated (Wells and Delhanty,
2001). Alternatively, analysis is performed on trophectoderm cells from the blastocyst stage at 5 days after fertilisation, when more cells can be harvested and thus a greater quantity of DNA can be extracted to provide
119 for more robust tests, although embryo survival up to this time point is difficult to guarantee. (Wells and Delhanty, 2001). Typically, PGD for monogenic disorders is performed on 1-2 single blastomeres that are biopsied at the cleavage stage (Wells and Delhanty, 2001). Data show that removal of these cells does not affect embryo survival (Hardy et al.,
1990).
PGD has been successfully used for the analysis of embryonic DNA material for various inherited genetic diseases such as cystic fibrosis
(Handyside et al., 1992), β-thalassemia (Kuliev et al., 1999; Jiao et al.,
2003), spinal muscular atrophy (Fallon et al., 1999), sickle cell disease
(Kuliev et al., 2001), and Marfan syndrome (Blaszczyk et al., 1998), usually via monoplex or multiplex PCR amplification of diagnostic disease markers using specific oligonucleotide primers to amplify genomic regions of interest, followed by mutation analysis methods such as heteroduplex analysis (Handyside et al., 1992; Ao et al., 1998), restriction endonuclease digestion (Kuliev et al., 1999), and DNA fragment size analysis to detect deletions and insertions (Sermon et al.,
1998). Alternatively, PGD can also be performed using genome hybridisation techniques such as FISH (Muggleton-Harris et al., 1995;
Veiga et al., 1999) and CGH (Malmgren et al., 2002).
The major technological hurdle for PGD is the inability to provide significant quantities of DNA for testing. The 3-7pg of DNA present in single haploid or diploid human cells requires many DNA amplification
120 cycles with PCR for it to be useful in any molecular analyses (Wells and
Delhanty, 2001). The large number of amplification cycles in a single cell PCR reaction can result in contamination or allelic dropout, which can possibly lead to misdiagnoses in up to 3% of cases (Drury et al.,
2001; Wells and Delhanty, 2001) (Figure 4.2). This has ethical implications due to the subsequent destruction of abnormal embryos, which is heavily regulated if not completely outlawed in countries such as Germany (Ludwig et al., 2001) and until recently in France (Wells and Delhanty, 2001).
Figure144.2. An illustration depicting the process of preimplantation genetic diagnosis (PGD). Briefly, in vitro fertilisation (IVF), in conjunction with intracytoplasmic sperm injection (ICSI), is required to generate an embryo. Embryo biopsy is performed at 3 days after fertilisation, whereby 1-2 single blastomeres are extracted. These single cells undergo many PCR amplification cycles to allow the DNA within the cells to be useful for the detection of any inherited mutations or disorders. However, the large number of cycles involved can introduce contamination and allelic dropout, resulting in misdiagnosis.
PCR-based whole genome amplification (WGA) methods (see Section
1.3.1) have been used in an attempt to overcome the problem of limiting
DNA samples in CGH and PGD analysis (Ao et al., 1998; Wells et al.,
121 1999; Voullaire et al., 1999; Tanabe et al., 2003; Jiao et al., 2003; Pirker et al., 2004). Although WGA protocols take longer to perform than conventional single cell multiplex PCR strategies, they provide a larger amount of DNA template for analysis, allowing the interrogation of a much greater number of loci and producing a resource of DNA that can be stored for future investigation of the same cell. However, no PCR- based WGA method as yet exists that is able to faithfully generate sufficient template material from limiting samples, to generate product with minimal amplification bias that is suitable for clinical diagnostic assays such as PGD and CGH. As described in Chapter I, PCR-based
WGA methods for amplifying low copy number DNA samples suffer from preferential amplification, allelic dropout and incomplete loci coverage. These factors result in significant bias of up to 1,000,000-fold between different loci within the same DNA sample (Dean et al., 2002).
Furthermore, the identification of false-positives and false-negatives, such as false gains at 1p and 16p or false losses at 4q resulting from
CGH of DOP-PCR products, can occur in PCR-based WGA products and contribute to possible misdiagnosis events (Pirker et al., 2004).
Although the isothermal multiple displacement amplification (MDA)
WGA method, which uses the highly processive and accurate φ29 DNA polymerase, possesses many advantages over PCR-based WGA methods as outlined in the previous chapters, this method still suffers up to 3-fold amplification bias at several loci along the human genome (Dean et al.,
2002; Hosono et al., 2003; Lage et al., 2003; refer to Chapter 1, Section
122 1.3.2.2). Although the degree of bias in MDA is much less than that encountered in PCR-based WGA methods, it is sufficient to cause the over- or under-representation of certain regions within the genome.
Hence, routine applications of MDA in diagnostic pathology laboratories have not been established, as methods for overcoming amplification bias in MDA have not been described. However, the potential utility of MDA in clinical diagnostics has been demonstrated for detecting gene rearrangements and chromosomal translocations (Luthra and Medeiros,
2004); PGD analysis of single cells via FISH and array-CGH (Hellani et al., 2004); and amplifying single sperms for gene copy number quantification, short tandem repeat (STR) and single nucleotide polymorphism (SNP) genotyping (Jiang et al., 2005).
This chapter presents the results from Aim III for examining the potential utility of MDA products in clinical diagnostics, specifically
CGH. An initial evaluation comprised an assessment of the capacity for high molecular weight products generated from MDA negative (no
DNA) controls to hybridise to human DNA sequences. An assessment was also performed on the utility of the GenomiPhi™ MDA system for amplifying limited DNA samples for downstream use in CGH to detect chromosomal abnormalities. Results in this section include the identification of regions that are susceptible to amplification bias by
GenomiPhi™ MDA and a novel but simple modification to the MDA reaction that reduced the degree of bias without compromising product yield and quality. The modified method allows uniform amplification of
123 limiting clinical samples by MDA and displays reduced amplification bias compared to the original unmodified protocol. The DNA template generated from this modified method is suitable for use in downstream
CGH, allowing disease diagnosis of single cell samples and PGD of single blastomeres, extracted from embryos generated by IVF. The large amount of MDA product synthesised from limited samples allows independent confirmatory testing to minimise the occurrence of misdiagnosis and permits further genetic analysis or sample archiving of precious samples.
4.2. Results and Discussion
As presented in Chapter 3, it was shown that MDA is sensitive down to the level of a single genome (or 6.4pg of genomic DNA) and consistently yielded approximately 5µg of DNA product suitable for use in downstream genetic analyses. However, in this section of the project, a higher concentration of DNA template was used throughout the analysis. DNA template originated from a phenotypically normal human male extracted from a whole blood sample. The absence of any cytogenetic anomalies was confirmed by CGH of 1µg unamplified genomic DNA as shown in Figure 4.3, which showed normal (1:1) dosage of chromosomal material. The genomic DNA sample was diluted to 1ng/µL (approximately 156 genomic equivalents) for use in
124 GenomiPhi™ MDA and subsequent analysis by CGH to evaluate the utility of MDA products in downstream clinical diagnostic applications.
Figure154.3. CGH profile for a normal human male genomic DNA sample. CGH results show a phenotypically normal human male test sample, with ratios between test (green) and reference (red) fluorescence levels close to 1:1 (black central line in each profile). Minor exceptions are observed in the centromeres of some chromosomes, such as chromosomes 1, 3, 9, but this is due to incomplete suppression using human Cot1 DNA to minimise cross- hybridisation between repetitive DNA regions.
Human male genomic DNA at a concentration of 1ng/µL, rather than single human cells, was used as the DNA template for MDA and downstream CGH testing as this sample was not limiting. Nevertheless, a clinical sample with a DNA concentration of 1ng/µL could still be considered as a limiting specimen that would potentially prohibit CGH
125 analysis without the inclusion of a WGA step. It should be noted that for the purposes of Aim III, DNA from a male donor was preferred over a female donor, as a male sample allows the interrogation of both X and Y sex chromosomes, therefore allowing the interrogation of all human chromosomes via CGH.
4.2.1. Assessment on the possibility of misdiagnosis caused by MDA products from a negative (no DNA) reaction control
As described in Chapter 3, the production of high molecular weight
DNA was observed in MDA negative control reactions that did not contain any DNA template, present as a high molecular weight DNA smear on agarose gels that appeared similar to that observed in MDA positive controls and MDA reactions containing input DNA template
(see Figure 3.1.B). In order to ensure that non-specific MDA product was not a possible source of misdiagnosis, CGH was performed using purified product DNA from an MDA negative control (no DNA) reaction. Equal amounts of non-specific MDA product labelled with the
SpectrumGreen™ fluorochrome and normal human male reference DNA labelled with SpectrumRed™ was precipitated simultaneously and allowed to hybridise to immobilised normal human metaphase chromosomes, in the presence of human Cot1 DNA to prevent cross- hybridisation of repetitive DNA sequences.
126
Figure164.4. CGH analysis of non-specific MDA products. Non-specific MDA products (labelled with the green SpectrumGreen™ fluorochrome, top left) did not hybridise on to human metaphase chromosomes, as indicated by the lack of fluorescence. Contrast this to the strong fluorescence exhibited by the reference DNA (labelled with the red SpectrumRed™ fluorochrome, bottom left). Absence of fluorescence from the green channel creates an erratic CGH profile (right panel).
Labelled non-specific MDA products did not appear to hybridise to normal human metaphase chromosomes, as indicated by the absence of green fluorescence along the arms of all chromosomes, suggesting the low possibility of misdiagnosis from non-specific MDA amplification products (Figure 4.4). The faint appearance of the metaphase chromosomes required an increase in illuminance and brightness to enable photographs of the slide to be taken. This is in contrast to the intense red fluorescence displayed by the labelled reference DNA.
Despite the lack of data from the green channel, combined analysis using the CytoVision 2.77 software (Applied Imaging, San Jose, CA, USA) could be performed and resulted in an erratic CGH profile. It appears
127 that this is a limitation of the analysis software, which attempted to generate a CGH profile despite incomplete fluorescence data.
Non-specific amplification product that is generated in MDA negative
(no DNA) controls is hypothesised to consist of hyperbranched primer concatemer arrangements or primer-primer duplexes that form in the absence of DNA template, as described in Chapter 3.
4.2.2. Amplification bias in MDA-amplified normal human genomic DNA
Due to its genome-wide approach, CGH was chosen as the best tool for detecting any chromosomal regions that are susceptible to amplification bias by MDA. It was hypothesised that regions of bias resulting from
MDA of a cytogenetically normal DNA sample will visually manifest as artificial imbalances. For these purposes, normal male genomic DNA sample was subjected to MDA for use in downstream CGH.
Comparisons were made between MDA-amplified and unamplified
DNA samples.
Following the manufacturer’s methods (see Section 2.5.1), amplifying
1ng of normal male genomic DNA in a 20µL GenomiPhi™ MDA reaction consistently generated 5µg of product DNA, as observed previously (see Section 3.2.1, Table 3.1). Prior to CGH, the MDA product was diluted to 1:200 with ET buffer and amplified by PCR, using at least two primer sets for human DNA sequences located on
128 different chromosomes, such as TYR (11q14.13), CITED1 (Xq11), and
TYRP1 (9p23), followed by detection of PCR amplicons using agarose gel electrophoresis to ensure the presence of product DNA template, thus indicating successful MDA. Ethanol purification and quantitation of the
MDA products by A260/A280 spectrophotometry were required prior to
CGH analysis. CGH involved hybridising equal amounts of
SpectrumGreen™-labelled sample MDA product and SpectrumRed™- labelled male reference DNA in the presence or absence of human Cot1
DNA. The respective CGH profiles are shown in Figure 4.5.
129
(a) (b)
Figure174.5. CGH profiles for MDA-amplified normal human male genomic DNA (a) with Cot1 suppression and (b) without Cot1 suppression for repetitive human DNA sequences. Without Cot1 suppression, CGH reveals reduced telomeric and centromeric signals after MDA, indicating loss of some repetitive sequences in these chromosomal regions during the MDA reaction. However, both CGH profiles indicate the presence of imbalances (aneuploidy) in MDA-amplified 01-0124 product that were not observable in the CGH profile for the original unamplified 01-0124 sample (Figure 4.3). These artificial imbalances suggest the presence of amplification bias resulting from the MDA reaction.
130 Reduced telomeric and centromeric signals were observed in the CGH profile for MDA-amplified DNA without human Cot1 DNA suppression
(Figure 4.5.b). The reduction of telomeric and centromeric signals suggests that some loss of repetitive sequences within these chromosomal regions occurred during MDA using the GenomiPhi™ system. This result supports a previous observation made by Dean et al.
(2002). When comparing the results from CGH analysis with and without Cot1 suppression, the most notable loss of telomeric and centromeric signals appear in chromosomes 1, 2, 3, 4, 9, 11, 12, 19, 20 and Y.
When comparisons were made between the CGH profiles for the unamplified genomic DNA sample (Figure 4.3) and the MDA-amplified sample (Figure 4.5), it is evident that several chromosomal regions deviate from the normal 1:1 ratio after performing MDA, suggesting localised amplification bias at these sites. Regions of bias were consistent and reproducible when tests were replicated, with or without
Cot1 suppression. It appeared that the observed bias was not random, with specific regions appearing to be more susceptible to bias compared to other regions (Figure 4.6). MDA amplification bias was present as both over- and under-amplification (Figure 4.7), with some regions becoming over-represented by up to 3-fold as determined by CGH.
These results support observations previously reported in the literature, which identified the enrichment of numerous loci by up to 225% after
131 MDA of a normal genomic DNA sample (Dean et al., 2002; Hosono et al., 2003; Lage et al., 2003).
Figure184.6. Non-random distribution of amplification bias. After MDA, specific chromosomal regions consistently appeared to be more susceptible to amplification bias compared to other regions. Regions appearing as green indicate over-representation of DNA material, whereas regions appearing as red indicate under-representation or loss of DNA material. Some regions display uniform green-red fluorescence, indicating equal representation of genomic material (close to 1:1) between MDA-amplified and unamplified samples. Compare this figure to the CGH profiles in Figure 4.5.
Figure194.7. Chromosomal regions affected by MDA amplification bias. Over-amplified regions are coloured green, whereas under-amplified regions are red. White regions indicate regions where bias was unobserved, or where representation ratios were close to 1:1 between MDA-amplified and unamplified samples.
132 Certain chromosomes were found to consistently display distinct patterns of over- and under-amplified regions, such as those observed on chromosomes 1, 6, 11 and 12, which could be readily identified under the microscope at 100x magnification with oil immersion. Reducing the
MDA reaction time from 18 hours to 6 hours did not inherently alter or reduce the appearance of bias (data not shown). However, a reduction in fluorescence intensity was noticed at 6 hours, possibly caused by lower final yields. Nonetheless, amplification bias is clearly not the result of prolonged incubation of the MDA reaction. An 18-hour MDA reaction is preferable over a 6-hour reaction, primarily because of greater product
DNA yields.
Due to the non-random distribution of amplification bias present in
MDA-amplified DNA template, it was hypothesised that unique characteristics were possibly shared within regions of both over- and under-amplification. The chromosomal region 6p21-p22 is known to contain a high concentration of genes, specifically human leukocyte antigens (HLA) of the major histocompatibility complex as well as other genes of clinical and metabolic significance as viewable on Ensembl
(http://www.ensembl.org) and the Online Mendelian Inheritance in Man
(OMIM; http://www.ncbi.nlm.nih.gov/omim) websites. From a graphical representation for the distribution of known genes on chromosome 6 obtained from Ensembl (Figure 4.8, right panel, black bars), it was apparent that the region of MDA over-amplification (up to 2-fold) on
6p21 (Figure 4.8, left panel) correlates to a region of very high gene
133 density, whereas the occurrence of under-amplification correlates to sites of lower gene density.
Figure204.8. Correlation between a region of MDA over-amplification on chromosome 6 to a region containing a high concentration of known genes. Chromosomal region 6p21, which exhibits up to 2-fold over-amplification bias after MDA as indicated by CGH (left panel), appears to contain a high density of known genes as indicated by the left-most histogram (black bars) in a graphical representation of gene distribution along chromosome 6 (right panel), obtained from Ensembl (Hubbard et al., 2005). In contrast, under-amplification at 6q appears to correlate with regions of lower gene concentration.
Based on observations presented in figure 4.8, regions of the human genome that are over-represented post-MDA appear to be primarily localised in regions of high gene density, which contain unique DNA sequences. In contrast, under-represented regions are attributed to sites within the human genome where gene density is low or those that contain a high number of repetitive DNA sequences. Similar observations were made for other chromosomes that displayed significant amplification bias (Figure 4.9).
134
(a)
(b)
(c)
Figure214.9. Correlation between other regions of MDA amplification bias to locations of high or low gene density. Other chromosomal regions that are over- or under-amplified by MDA as determined by CGH appear to contain high or low concentrations of genes respectively, as indicated by CGH profiles and chromosomal ideograms obtained from Ensembl (Hubbard et al., 2005) for: (a) chromosome 1; (b) chromosome 11; and (c) chromosome 12.
135 It was difficult to perform direct comparisons for any similarities in biased regions identified in this study with previously published reports.
Difficulties exist due to the use of different assays to identify regions exhibiting bias, such as TaqMan analysis (Dean et al., 2002; Hosono et al., 2003), CGH (Dean et al., 2002) and cDNA array-CGH (Lage et al.,
2003), and the limited amount of data presented in these reports. Results from previous studies, specifically those that utilised TaqMan analysis and cDNA array-CGH, were obtained from the interrogation of known and specific loci or regions of interest. Data from these reports do not appear to cover the whole human genome, unlike the holistic approach taken in this study. Furthermore, CGH results from Dean et al. (2002) are inadequate to warrant a full cross comparison of data, as the authors focused on centromeric and telomeric sites for general fluorescence uniformity along chromosomal arms. An assessment of the available literature did not identify any recent publications on MDA amplification bias or the detailing of specific regions that exhibit amplification bias post-MDA.
However, recent publications of the draft human DNA sequence
(International Human Genome Sequencing Consortium, 2001; Venter et al., 2001) have confirmed the over-abundance of repetitive DNA sequences within the human genome. Even though sequencing was primarily focused on euchromatic (GC-rich, gene-rich) regions of the genome, only 5% of sequences were identified as unique and protein- coding, while greater than 45% of sequences were classed as repetitive
136 DNA. Heterochromatic regions, which are assumed to be gene deficient, are generally ignored in sequencing projects due to cloning and sequencing difficulties. Some euchromatic regions were found to be nearly devoid of repeats, possibly due to the presence of large-scale cis- regulatory elements that cannot tolerate interruption by long stretches of repetitive DNA, such as the homeobox (HOX) gene clusters that are located on various chromosomes. It is interesting to note that the sequence near 1p36 consists of only 5% repetitive DNA, and in this study was found to exhibit at least 2-fold over-representation after MDA
(Figure 4.9.a). Furthermore, the largely heterochromatic Y chromosome, consisting mainly of repetitive DNA with only a small number of gene- coding DNA sequences (Bachtrog and Charlesworth, 2001; Willard,
2003), exhibited under-representation within the long (q) arm (see figures 4.5 and 4.7).
Observations up to this point led to the development of theories for methods that might ensure successful MDA of gene-poor regions, at a similar rate to the amplification of gene-rich regions, without inducing amplification bias. It was thought that the random hexamers used in the
GenomiPhi™ MDA system were selective for gene-rich regions, ensuring that these sites would have a greater chance of being successfully amplified compared to gene-poor regions. It was hypothesised that the addition of more hexamers or primers specifically designed for gene-poor sites to the GenomiPhi™ reaction might alleviate amplification bias. However, it was possible that the addition of
137 additional reagents to the GenomiPhi™ reaction would alter the concentration of buffers and salts due to a change in reaction volume, thereby potentially changing the GenomiPhi™ reaction conditions and hence affecting MDA reaction kinetics.
An alternative method involved making slight changes to the MDA reaction protocol. Dean et al. (2002) found that omitting the step for denaturing template DNA produced more uniform amplification by
MDA, but supporting data to show this were not presented. It is known that in vivo partial separation or denaturation of double-stranded DNA
(dsDNA) is necessary for replication and transcription, and is caused by several proteins that use ATP to break the hydrogen bonds connecting the two DNA strands (Voet et al., 1999). Denaturation of dsDNA in vitro is usually performed using heat; or in alkaline conditions (elevated pH), which causes a change in the charge differential of the side groups that are involved in non-covalent bonding between the bases; or by using chemical compounds such as urea and formamide to denature the DNA by reacting directly with the bases, thus preventing normal base-pairing.
Re-hybridisation of denatured dsDNA is accomplished by returning the
DNA to conditions that promote dsDNA formation.
Complementary base pairs within dsDNA have also been shown to frequently open and “breathe” because of natural thermal motion of the
DNA strand, with each base pair remaining bonded for only 10-2 seconds, and each breathing event lasting for approximately 10-7 seconds
138 (Guéron et al., 1987). During natural breathing of dsDNA, highly repetitive DNA sequences re-anneal to their complementary strands more rapidly than unique regions (Wetmur and Davidson, 1968; Guéron et al., 1987). It was therefore proposed that without a thermal denaturation step at 95°C for 3 minutes, repetitive sequences within the
DNA template would possess sufficient natural capacity to allow annealing of random hexamers to both gene-rich and gene-poor regions.
Comparisons were thus made between CGH profiles from MDA reactions where the denaturation step was either included or omitted
(Figure 4.10). Regions of amplification bias were still apparent in MDA reactions with and without a denaturation step. However, CGH profiles for denatured and non-denatured GenomiPhi™ MDA products were almost mirror images of each other (Figure 4.10). A region that was over-amplified in the denatured profile was generally under-amplified in the non-denatured profile. For example, region 1p36 exhibited over- amplification in the denatured reaction, but displayed under- amplification in the non-denatured reaction. Similar observations were made on other chromosomes, such as 6, 11, and 12. Regions exhibiting amplification bias were identical to those identified earlier. However, the degree of bias was not as pronounced as observed previously. This was attributed to a new GenomiPhi™ kit that was used in this particular experiment.
139
(a) MDA (+ denaturation step) (b) MDA (- denaturation step)
Figure224.10. CGH profiles for MDA reactions where the denaturation step (95°C for 3 minutes) for denaturing sample DNA was either (a) included or (b) omitted. Although not as extreme as previous observations, regions of amplification bias are still present in both profiles. However, the two CGH profiles appear as reversed images of each other. Essentially, a region that is over-amplified in the denatured reaction generally appears to be under-amplified in the non-denatured reaction.
140 Generally, the denaturation of template DNA prior to MDA promoted over-amplification of gene-rich regions, whereas omitting the denaturation step allowed over-amplification of gene-poor regions.
Based on these results, it was hypothesised that the combination of an equimolar amount of product from denatured and non-denatured MDA reactions, or simply combining denatured and non-denatured DNA template prior to MDA, would nullify any amplification bias. Equimolar mixing of denatured and non-denatured DNA was achieved by mixing the MDA products that were used to generate the CGH profiles in Figure
4.10 at a 1:1 ratio (Figure 4.11.a). Alternatively, mixing can be achieved prior to MDA (Figure 4.11.b). This was performed by adding 1µL of
DNA to 9µL of GenomiPhi™ Sample Buffer, and dividing the 10µL mixture into two 5µL portions. Only one 5µL portion was subjected to denaturation at 95°C for 3 minutes and rapidly cooled on ice before mixing the two portions and adding φ29 DNA polymerase. Both procedures have advantages and disadvantages that should be taken into account when applying these methods for routine practical use. The second method requires more steps and involves some sample manipulation and tube transfers, and is therefore more prone to error and contamination, but only 1µL of sample is consumed. In contrast, the first method requires two MDA reactions to be performed: one reaction incorporates a denaturation step (as per the manufacturer’s instructions) and a second reaction has the denaturation step omitted. This requires twice the amount of DNA sample and MDA reagents compared to the second method. Results from this experiment are shown in Figure 4.12.
141
(a) Method I
(b) Method II
Figure234.11. Methods used for mixing denatured and non-denatured MDA products or DNA templates. (a) Method I requires two individual MDA reactions, with only one incorporating a denaturation step. Purified MDA products are then combined at equal amounts for CGH analysis. (b) Method II involves dividing the DNA sample into two equal portions, and only subjecting one portion to denaturation, prior to re-mixing of both portions and addition of φ29 DNA polymerase for MDA. The purified MDA product is then subjected to CGH.
142 (a) MDA “denatured” + MDA “non-denatured” (Method I)
(b) DNA “denatured” + “non-denatured” prior to MDA (Method II)
Figure244.12. CGH profiles from MDA products that were generated using modified GenomiPhi™ method I or II. Regions that previously exhibited over- and under-amplification in the individual denatured/non-denatured reactions were significantly nullified upon mixing of the (a) denatured/non- denatured MDA products (Method I) or (b) denatured/non-denatured DNA prior to MDA (Method II).
143 The degree of bias that was previously observable, such as that on chromosomes 1, 6, 11 and 12, was significantly reduced after performing either of the two modified MDA methods: (a) mixing denatured and non-denatured MDA products, or (b) mixing denatured and non- denatured DNA prior to MDA, as determined by CGH (Figure 4.12).
The CGH profiles indicate normal (1:1) DNA dosage for most chromosomal regions that previously exhibited either over- or under- representation. Exceptions included heterochromatic regions containing an over-abundance of repetitive DNA sequences, such as the centromeres and p-arms of acrocentric chromosomes and the repeat-rich q-arm of chromosome Y.
However, the acrocentric chromosomes (chromosomes 13, 14, 15, 21,
22) are known to contain 73% of all human alpha repetitive satellite
DNA sequences (Choo et al., 1988) and the nucleolar organiser regions consisting of human ribosomal gene repeats (Sullivan et al., 2001).
Much of the human Y chromosome is also composed of repetitive satellite DNA, the bulk of which is located in the large heterochromatin region (roughly 30mb in size) along the q-arm, which consists of Alu and LINE repetitive elements (Bachtrog and Charlesworth, 2001), as well as Y-specific repeats such as 3.4kb DYZ1 and 2.5kb DYZ2 repeat families that account for up to 70% of the DNA content in the Y chromosome (Cooke et al., 1983). Under-amplification near the centromere of chromosome 9 also could not be overcome by the modified MDA methods. However, it is known that this region of
144 chromosome 9 contains the largest autosomal block of heterochromatin in the human genome (Humphray et al., 2004). The loss of some telomeric and centromeric regions also appears to be outside the control of the modified MDA methods.
4.3. Summary and Conclusions
MDA reactions that do not contain input DNA template were found to generate high molecular weight non-specific amplification products that were similar in yield and appearance to other MDA reactions containing
DNA sample. However, the non-specific amplification product did not perform in downstream applications such as PCR using human-specific oligonucleotide primers or comparative genomic hybridisation to human metaphase chromosomes. Non-specific MDA products therefore do not appear to interfere with human diagnostic applications. Nevertheless, the inclusion of an MDA negative (no DNA) control should be mandatory in order to monitor for the occurrence of contamination. Likewise, MDA positive controls that contain a known amount of DNA are essential for monitoring the success of the MDA reaction.
Using the genome-wide approach of comparative genomic hybridisation
(CGH), the three-fold amplification bias present in MDA-amplified products that was previously reported by Dean et al. (2002), Hosono et al. (2003) and Lage et al. (2003) was confirmed in this study. Over-
145 amplification was localised to regions of high gene concentration, containing unique DNA sequences, whereas under-amplification was located in regions containing a high number of repetitive DNA sequences and low gene concentration. Omitting the denaturation step in the GenomiPhi™ MDA protocol was postulated to allow better primer binding to repetitive sequences in regions of lower gene density, and led to the development of modified GenomiPhi™ protocols. The modified
MDA protocols, based on mixing denatured and non-denatured MDA products or denatured and non-denatured genomic DNA prior to MDA, were shown to dramatically reduce amplification bias in most chromosomes. However, under-amplification of heterochromatic regions, specifically those on acrocentric chromosomes, and chromosomes 9 and Y, were not overcome by the modified methods.
This is attributed to the very high concentration of repetitive elements within these regions, causing possible amplification difficulties during
MDA.
The two modified GenomiPhi™ MDA methods may be suitable for specific situations, depending on the amount of sample that is available for testing. Method I, where MDA products are generated from two
MDA reactions (with and without a denaturation step), requires double the amount of DNA template compared to Method II, where a DNA sample is divided into two portions (with one portion undergoing denaturation and the other portion remaining non-denatured) prior to
146 incubation of the MDA reaction. If the DNA sample is non-limiting,
Method I should be performed.
Key findings that were highlighted in this chapter open the way for future research. The utility of MDA products for the diagnoses of genetic conditions in limited clinical samples should be further assessed.
The modified MDA methods described here could be applied to the amplification of human patient samples with known abnormalities, such as tumour/cancer cell lines including the chronic myelogenous leukaemia (CML) cell line K562, in order to determine if the known abnormality is conserved after MDA. Furthermore, the modified MDA method should be assessed for use in both PCR- and CGH-based genetic analysis of single cell samples, such as PGD of amniocytes or blastomeres, and testing of cancer cells and foetal DNA circulating in maternal plasma.
147 CHAPTER FIVE
FINAL SUMMARY AND CONCLUSIONS
Multiple displacement amplification (MDA) using the GenomiPhi™
DNA Amplification Kit (Amersham Biosciences, Piscataway, NJ, USA) was found to be a useful tool for amplifying low amounts of DNA material. As presented in Chapter 3, MDA was able to amplify the DNA present within a single human cell, or its equivalent of 6.4pg of genomic
DNA, and consistently generated 5µg of high molecular weight MDA product for every 20µL reaction. At these yields, MDA can potentially yield sufficient DNA for up to 20,000,000 single locus PCR assays.
MDA could also be applied to the amplification of a variety of simulated forensic samples, including used cigarette butts, licked stamps, touched objects, single shed hairs, finger swabs and buccal swabs that were extracted using simplified Chelex or buffer digest protocols. After MDA, simulated forensic samples were amplified by up to 200,000-fold compared to the original unamplified DNA sample, yielding sufficient
DNA for up to 2,000,000 single locus PCR assays.
148 Single cell samples could be subjected directly to MDA without any prior purification or lysis procedures. Simulated forensic samples only required brief purification using modified extraction protocols prior to
MDA. The modified protocols consisted of simplified 5% Chelex-100 and buffer digest protocols as outlined in Table 3.2. The low extraction volumes of 50µL and 20µL for the 5% Chelex and buffer digest protocols respectively help to concentrate the DNA recovered from low copy number forensic samples. This ensures that there is sufficient DNA per microliter of extract that is added to the MDA reaction. The Chelex resin also enables removal of any inhibitors in the DNA extract that may affect the success of MDA, and excessive inhibition can generally be overcome by diluting the DNA extract by 1:5 or 1:10 in ET buffer prior to MDA. These modified protocols are simple to perform with quick processing times as little as 50 minutes. Furthermore, the Chelex protocol is an established and validated forensic DNA extraction procedure and can therefore be performed by any forensic laboratory. In summary,
DNA extracts obtained through these protocols were suitable for use in
PCR and MDA.
MDA negative control reactions that did not contain any input DNA were also found to generate non-specific high molecular weight MDA products that were similar in yield to other reactions containing DNA material. According to the manufacturers, the synthesis of non-specific product in no-template MDA reactions is expected. It is presumed that the non-specific MDA products consist of primer-primer duplexes or
149 primer concatemers, the formation of which is promoted due to a lack of amplifiable DNA template. However, as discussed in chapters 3 and 4, the non-specific DNA material did not perform in downstream applications, including PCR assays using human-specific oligonucleotide primers and hybridisation to human metaphase chromosomes via comparative genomic hybridisation (CGH). As presented in Chapter 4, analysis of MDA negative control products using CGH did not indicate hybridisation of the non-specific products to human DNA sequences. It is therefore unlikely that non-specific MDA products would interfere in human diagnostic applications.
MDA products could be used in downstream PCR-based assays without any prior purification. The ability to bypass a purification step allows direct use of the MDA products in downstream assays, therefore simplifying sample processing and preventing the occurrence of excessive sample handling or tube transfers, thus conserving sample integrity and also enabling automated procedures. However, purification was required for quantitation of the MDA products by
spectrophotometric absorption (A260/A280, with background correction at
320nm) in order to remove protein components and residual primers, which would otherwise contribute to incorrect quantification data.
Purification of MDA products should also be performed for downstream genome hybridisation assays such as CGH, as residual protein components may affect resolution of the assay due to excessive background noise.
150
This study identified a dependence of MDA on DNA material of a sufficient quality with fragments greater than 200bp in length. MDA reactions containing degraded DNA fragments less than 200bp were found to generate high molecular weight DNA product, but could not be utilised in downstream PCR-based applications. This is presumably because the degraded DNA fragments at less than 200bp in length are too short to allow multiple random hexamers to bind and maintain MDA reaction kinetics. Hypothetically, if primers bind close to the middle of the fragments and are extended by φ29 DNA polymerase, each successive strand displacement event would generate shorter templates until primer binding and extension cannot proceed. Due to a lack of amplifiable DNA, the use of degraded DNA material in MDA would also promote the formation of non-specific products. In order to ensure successful MDA of precious or limited samples, DNA extracts should be screened for fragments greater than 200bp. Screening a sample for DNA templates that are compatible for amplification by MDA can increase the efficient use of that sample. In cases where precious or limited forensic
DNA samples are highly fragmented, the specimens should be utilised in assays that can maximise the amount of data obtainable from those samples. Highly degraded forensic samples can be amplified using a commercial or in-house miniSTR multiplex system as an alternative to the conventional STR analysis. MiniSTR systems utilise modified primers that allow amplification of shorter target STR regions by PCR and is therefore suitable for amplifying degraded DNA samples. In the
151 case of freshly obtained single cell samples for use in clinical diagnostic applications, screening for degraded DNA templates is not required.
Proposed guidelines for the use of MDA in forensic biology were presented in Section 3.3.1.
For utility as a DNA sample archiving tool, MDA was able to amplify single bloodspot samples that were extracted using the 5% Chelex protocol. However, at 200µL extraction volumes, removal of heme and other blood components did not appear to be efficient. This resulted in inhibition of MDA, indicated by reduced yields or complete failure of the reactions. However, dilution of the blood extracts by 1:10 in ET buffer appeared to overcome the inhibitory effect. Subjecting 1µL of diluted blood extract to MDA was found to generate sufficient DNA template for up to 4,000 PCR assays. It is postulated that more DNA can be synthesised using scalable MDA reactions for the purposes of generating bulk amounts of DNA material. Guidelines for using MDA as a DNA sample archiving tool were presented in Section 3.3.2.
Overall, the findings from Chapter 3 suggest that MDA results in a higher yield in comparison to DOP-PCR and PEP. A large number of
PCR reactions are possible from as little as two individual cells after
MDA, enabling repeated rounds of retesting and archiving of limited and precious samples for future analysis. MDA allows a limiting DNA sample to be subjected to a variety of downstream applications in order to increase the amount of information obtainable from that sample. This
152 is in contrast to subjecting a limiting sample, in the order of one or two single cells, to a particular assay that can only be performed once due to limited sample availability. Any chance of obtaining data from that sample can be significantly compromised in the event of a reaction failure. The large amount of DNA template produced by MDA is also advantageous as it provides sample consistency. A number of different assays can be performed on one single sample source, without the need to re-extract a sub-sample for each application.
Chapter 4 highlighted the presence of approximately 3-fold amplification bias in MDA-amplified DNA templates, identified via the methodology of CGH. This finding supports previous observations as published in the literature (Dean et al., 2002; Hosono et al., 2003; Lage et al., 2003). Over-amplification was found to be localised in regions of high gene density that contain unique gene-coding sequences, whereas under-amplification was prevalent in regions of lower gene density that contain a high concentration of repetitive DNA sequences. It was postulated that the omission of the denaturation step in the GenomiPhi™
MDA protocol would allow better primer binding to repetitive DNA sequences. Modifications to the GenomiPhi™ protocol, based on mixing denatured and non-denatured DNA samples or MDA products, were found to significantly reduce the degree of bias in most chromosomes.
However, these modified procedures could not overcome the under- amplification of highly repetitive DNA sequences that are present along several chromosomes, including the centromeres and p-arms of
153 acrocentric chromosomes (chromosomes 13, 14, 15, 21, 22), the heterochromatic region of chromosome 9 and the highly repetitive region of Yq. Nevertheless, this improvement opens the way for the utility of MDA in clinical diagnostic applications that depend on uniform DNA templates, such as copy number analysis and gene dosage studies.
Future studies could be performed to build on the outcomes presented in this thesis. The novel modification to the GenomiPhi™ MDA procedure should be further assessed for use in PGD and genetic testing of a variety of single cell samples, such as amniocytes, blastomeres, cancer cell lines and foetal DNA circulating in maternal plasma. A cell line with known genetic abnormalities could be amplified by the modified MDA method to examine if the known abnormality is conserved after MDA, which would indicate the synthesis of highly uniform DNA products. An investigation into the use of the modified MDA method for amplifying limited forensic samples consisting of DNA mixtures, such as those obtained from sexual assault stains, followed by conventional STR analysis can also be performed in order to assess the conservation of allelic mixture ratios. Furthermore, a thorough assessment on allelic dropout in low copy number samples after MDA can also be performed using conventional STR analysis.
In summary, the findings presented in this thesis provide a valuable insight into the utility of MDA for applications in forensic biology and
154 clinical diagnostics. A proper understanding of the MDA system can lead to further improvements that would allow amplification of low copy number DNA samples to become more efficient and reliable. The use of
MDA in forensic biology applications can potentially improve the utility of low copy number DNA samples that previously could not be utilised in forensic investigations. In addition, the ability to utilise single cells as samples for diagnostic pathology tests may allow the development of less invasive sample collection methods, thereby improving patient-care.
Furthermore, the ability to essentially generate an unlimited supply of
DNA via MDA does not restrict the amount of tests and applications that can be performed by scientists performing research in an endless number of disciplines.
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