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STR Analysis NEAFS CE-DNA Workshop (Butler and McCord) Sept 29-30, 2004 Capillary Electrophoresis Outline for Workshop in DNA Analysis • Introductions •STR Analysis STR Analysis • Introduction to CE and ABI 310 NEAFS Workshop • Data Interpretation Mystic, CT • Additional Topics – Real-time PCR and miniSTRs September 29-30, 2004 • Higher Throughput Approaches Dr. John M. Butler • Troubleshooting the ABI 310 (Participant Roundtable) Dr. Bruce R. McCord • Additional Topics – Y-STRs, validation, accuracy • Review and Test Training Materials on STRBase http://www.cstl.nist.gov/biotech/strbasehttp://www.cstl.nist.gov/biotech/strbase Historical Perspective on DNA Typing Human Identity Testing Identifiler 5-dye kit US National and ABI 3100 Database (NDIS) 2002 • Forensic cases -- matching suspect with launched evidence UK National CODIS loci PowerPlex® 16 Database launched defined (16 loci in single amp) • Paternity testing -- identifying father 2000 1998 • Historical investigations FSS STR typing with Quadruplex CE is fairly routine • Missing persons investigations 1994 1996 First STRs First commercial • Mass disasters -- putting pieces back together developed fluorescent STR multiplexes 1990 • Military DNA “dog tag” 1992 Capillary electrophoresis • Convicted felon DNA databases of STRs first described 1985 PCR developed http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 1 NEAFS CE-DNA Workshop (Butler and McCord) Sept 29-30, 2004 National DNA Index System (NDIS) Analogy of Computers and DNA Typing • Computers originated in 1946 and began as sophisticated devices that were appreciated and utilized by a select few (the “nerds”). • Computer use has now expanded to the layperson and general household http://www.fbi.gov/hq/lab/codis/index1.htm use as computers have become easier to use. • One of the greatest advances for the personal computer was the advent of multitasking (the ability to perform simultaneous applications). Combined DNA Index System (CODIS) • PCR and DNA typing methods first began as experimental exercises in 1984 but have progressed to become a general workhorse among forensic Launched in October 1998 and now links all 50 states laboratories around the world. Used for linking serial crimes and unsolved cases with repeat offenders • Multiplexing is a corresponding breakthrough for PCR as simultaneous Convicted offender and forensic case samples along with a missing amplification of different segments of DNA can be performed in a single persons index reaction. Requires 13 core STR markers • STR typing is now widely used because of the availability of commercial kits >18,000 investigations aided nationwide as of June 2004 that makes DNA testing easier to perform. Contains more than 1.8 million DNA profiles Adapted from John J. Sninsky, PCR Applications: Protocols for Functional Genomics, Academic Press, 1999. Historical Comparison with Computers Importance of Standardized Data Formats GHz computing speeds 2002 • DNA databases work because everyone is Microsoft Windows 95 released submitting data on the same genetic markers (enabling multi-tasking) (6 SGM loci or 13 CODIS STR loci) to insure 2000 1998 Internet use is “communication” between all the samples Pentium chip fairly routine developed • Internet works because computers are 1994 1996 networked with common protocols (TCP/IP) World Wide 386, 486 Web and “speak” a common language 1990 1992 1985 First version of Windows software; Microsoft Corporation goes public Steps in DNA Analysis Collection Slot Blot 1 ng 0.3 ng Specimen Storage No DNA 0.5 ng •Report published in Nov 2000 0.5 ng 0.7 ng Extraction 1 ng •Asked to estimate where DNA Blood Stain Buccal swab 1 ng testing would be 2, 5, and 10 years Sample Collection DNA DNA Quantitation into the future & Storage Extraction Quantitation Conclusions PCR Amplification STR typing is here to Genotyping stay for a few years Multiplex PCR Amplification because of DNA Interpretation databases of Results STR Typing Database Male: 13,14-15,16-12,13-10,13-15,16 Storage & Searching http://www.ojp.usdoj.gov/nij/pubs-sum/183697.htm Interpretation of Results DNA Database http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 2 NEAFS CE-DNA Workshop (Butler and McCord) Sept 29-30, 2004 Basic Concepts DNA Amplification with the Polymerase Chain Reaction (PCR) PCR (polymerase chain reaction) – method of Components of PCR 5’ 3’ Thermal stable polymerase amplifying a specific region of the genome – go from 1 to 5’ 3’ dNTPs (building blocks) Starting DNA over a billion copies in 2-3 hours MgCl2 Template Buffer 3’ 5’ Primers (specific to site) 3’ 5’ Locus region of the genome being examined Separate strands (denature) Allele the state of the genetic variation being examined (STRs = number of repeat units) Forward primer 5’ 3’ 5’ 3’ (SNPs = base sequence at the site) Make copies Add primers (extend primers) 5’ 3’ 5’ (anneal) 3’ Chromosomes are paired so… Reverse primer Homozygous – Alleles are identical on each chromosome Heterozygous - Alleles differ on each on each chromosome InIn 3232 cycles cycles atat 100% 100% efficiency,efficiency, 1.071.07 billion billion copiescopies of of targetedtargeted DNADNA regionregion areare createdcreated PCR Process 5’ 3’ Starting DNA Template Thermal Cycling Parameters 80-500 bases 3’ 5’ Step in Protocol AmpFlSTR® kits PowerPlex® STR kits (Applied Biosystems) (Promega Corporation) Separate Initial Incubation 95 oC for 11 minutes 95 oC for 11 minutes Forward Primer strands (denature) a 5’ 3’ 5’ 3’ Thermal Cycling 28 cycles 30 cycles Denature 94 oC for 1 minute 94 oC for 30 seconds (cycle 1-10) o Add primers 90 C for 30 seconds (cycle 11-30) 5’ Anneal o o 3’ 5’ (anneal) 3’ 59 C for 1 minute 60 C for 30 seconds Reverse Primer Extend 72 oC for 1 minute 70 oC for 45 seconds Make copies (extend primers) Final Extension 60 oC for 45 minutes 60 oC for 30 minutes Final Soak 25 oC 4 oC Repeat Cycle, (until samples removed) (until samples removed) Copying DNA Exponentially a) The first 10 cycles are run with a denaturation temperature of 94 oC and the last 20 cycles are run at 90 oC instead. The Promega PowerPlex 1.1 and 2.1 kits also use specific ramp times between the different temperature steps that differ from the conventional 1 oC/second. Butler, J.M. (2001) Forensic DNA Typing, Figure 4.2, ©Academic Press Butler, J.M. (2001) Forensic DNA Typing, Table 4.3, ©Academic Press Advantages of PCR Potential Pitfalls of PCR • Minute amounts of DNA template may be used from as little as a single cell. • The target DNA template may not amplify due to the presence of PCR inhibitors in the extracted DNA • DNA degraded to fragments only a few hundred base pairs in length can serve as effective templates for amplification. • Amplification may fail due to sequence changes in • Large numbers of copies of specific DNA sequences the primer binding region of the genomic DNA can be amplified simultaneously with multiplex PCR template reactions. • Contaminant DNA, such as fungal and bacterial • Contamination from other human DNA sources sources, will not amplify because human-specific besides the forensic evidence at hand or previously primers are used. amplified DNA samples is possible without careful • Commercial kits are now available for easy PCR laboratory technique and validated protocols reaction setup and amplification. Butler, J.M. (2001) Forensic DNA Typing, Chapter 4, p. 50, ©Academic Press Butler, J.M. (2001) Forensic DNA Typing, Chapter 4, pp. 50-51, ©Academic Press http://www.cstl.nist.gov/biotech/strbase/NISTpub.htm 3 NEAFS CE-DNA Workshop (Butler and McCord) Sept 29-30, 2004 Tips for Avoiding Contamination DNA in the Cell • Pre- and post-PCR sample processing areas should be physically separated. • Equipment, such as pipettors, and reagents for setting up PCR chromosome should be kept separate from other lab supplies, especially 22 pairs + XX or XY those used for analysis of PCR products. • Disposable gloves should be worn and changed frequently. cell nucleus • Reactions may also be set up in a laminar flow hood, if available. • Aerosol-resistant pipet tips should be used and changed on Double stranded every new sample to prevent cross-contamination during liquid DNA molecule Target Region for PCR transfers. • Reagents should be carefully prepared to avoid the presence of ~3 billion total base pairs any contaminating DNA or nucleases. • Ultraviolet irradiation of laboratory PCR set-up space when the area is not in use and cleaning workspaces and instruments with isopropanol and/or 10% bleach solutions help to insure that Individual extraneous DNA molecules are destroyed prior to DNA nucleotides extraction or PCR set-up Butler, J.M. (2001) Forensic DNA Typing, Chapter 4, pp. 49-50, ©Academic Press Human Genome 23 Pairs of Chromosomes + mtDNA What Type of Genetic Variation? Located in cell nucleus Autosomes 2 copies Located in •Length Variation per cell mitochondria (multiple copies short tandem repeats (STRs) in cell cytoplasm) CTAGTCGT(GATA)(GATA)(GATA)GCGATCGT mtDNA 1 2 3 4 5 6 7 8 9 10 11 12 16,569 bp •Sequence Variation single nucleotide polymorphisms (SNPs) http://www.ncbi.nlm.nih.gov/genome/guide/ insertions/deletions Mitochondrial GCTAGTCGATGCTC(G/A)GCGTATGCTGTAGC 13 14 15 16 17 18 19 20 21 22 X Y DNA Sex- Nuclear DNA chromosomes 3.2 billion bp 100s of copies per cell Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 2.3, ©Elsevier Science/Academic Press Multiplex PCR Why STRs are Preferred Genetic Markers (Parallel Sample Processing) • Compatible primers are the key • Rapid processing is attainable to successful
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