The Polymerase Chain Reaction in Pathology

I Clin Pathol 1997;50:805-810 8050

Origins of . . J Clin Pathol: first published as 10.1136/jcp.50.10.805 on 1 October 1997. Downloaded from

The polymerase chain reaction in pathology

J J O'Leary, K Engels, M A Dada

A surprisingly simple method of making unlimited and transferred (blotting) from the gel to a copies of DNA fragments conceived under unlikely solid support (usually a nitrocellulose or nylon circumstances during a moonlit drive through the mountains of California. filter). The sequence of interest can then be Kary B Mullis' detected using a short fragment of DNA (oligonucleotide probe), which is complemen- tary to the DNA sequence of interest (hybridi- The movie J7urassic Park and real life events like sation). the 0 J Simpson trial have captured public Initially, radioactive labels (32P, 35S, 3H) were interest in molecular biology. The basis of used for probing, but later non-isotopic labels molecular biology is the understanding of the including biotin, digoxigenin, and fluorescein structure and function of DNA, RNA, and were employed. Three nucleic acid labelling proteins, as well as the techniques for manipu- methods are now described, including enzyme lating these molecules. In this review we dem- incorporation, chemical derivitisation, and onstrate how a single technique revolutionised chemical cross linking. The enzymatic labelling this research area and made molecular biologi- reactions for DNA include nick translation, cal methods accessible, not only to research random priming, and 5' and 3' end labelling groups but also to diagnostic pathology labora- using DNA polymerase, Klenow polymerase, tories. As a result, molecular pathology is now and Dnasel.5 For RNA detection, riboprobes firmly established as a pathology discipline, can be created using SP6, T3, and T7 in vitro providing new insights into the pathogenesis of enzymologies. Alternatively, synthetic oligonu- disease as well as innovative techniques in cleotides can be conveniently used as probes diagnosis. for DNA and RNA detection assays. Addition-

ally, PCR probe labelling methods, employing http://jcp.bmj.com/ Historical background biotin, digoxigenin or fluorescein labelled Although DNA was first isolated in 1869 by dNTPs in substituted molar ratios in the PCR Miescher, its double helix structure was not reaction mix, can also be used for generating described until 1953 by Watson and Crick.2 In probes for any hybridisation analysis. 1955, Arthur Kornberg of Stanford University A refinement of Southern blot analysis, of discovered DNA polymerase.' This cellular interest to pathologists, was first published in

enzyme is involved in DNA replication and 1969 by two groups working independently in on September 25, 2021 by guest. Protected copyright. repair by catalysing the addition of nucleotides the UK and the USA.67 The technique-in situ to the 3' end of an existing DNA chain. The hybridisation-allowed for the first time direct initiation of a new chain requires an existing correlation between hybridisation signals and oligonucleotide or polynucleotide chain, re- tissue morphology. The initial reports were fol- ferred to as a primer. The polymerase attaches lowed by application of the methodology to Department of nucleotides in a new DNA strand, complemen- cryostat, paraffin wax, chromosomal, and elec- Pathology, Cornell tary to nucleotides on corresponding positions tron microscopy preparations.81' Since then, University Medical of the parent DNA strand (template strand). numerous DNA and RNA targets have been College, New York, RNA polymerases are involved in the assembly demonstrated using both isotopic and non- USA of RNA from a DNA template (transcription). isotopic labels. J J O'Leary Over time, new tools for producing and Analogous techniques for RNA and proteins Department of manipulating DNA were developed. Restric- have been named northern and western Cellular Science, tion endonucleases (RE) cut DNA at specific blotting, respectively, as a play on the name University of Oxford, sequences (restriction sites), making it possible Southern. These techniques gained wide- Oxford, UK to isolate strands of DNA containing specific spread acceptance in the 1970s. K Engels M A Dada genes.3 In 1975, Edwin Southern described a The next significant breakthrough in mo- technique for the localisation of specific lecular biology was the development of rapid Correspondence to: sequences within genomic DNA by electro- DNA sequencing techniques. In the chemical Dr O'Leary, Department of phoretic transfer techniques.4 This technique, method of Maxem and Gilbert the sequence is Pathology, Cornell University Medical College, The New subsequently known as Southern blotting, determined from native DNA itself.'2 DNA is York Hospital, 1300 York involves the digestion of genomic DNA by one labelled at one end and then exposed to agents Avenue, New York, NY or more REs and the separation ofthe resulting that destroy one or two of the nucleotides 1002 1, USA. fragments by agarose gel electrophoresis. The resulting in fragments that can be separated Accepted for publication separated fragments of double stranded DNA and analysed by electrophoresis. The Sanger or 15 July 1997 are then separated into single stranded form dideoxy chain termination method parallels the 806 O'Leary, Engels, Dada

process of DNA replication.2 12 Starting with a disciplines such as cell biology, and medical primer, a DNA polymerase adds nucleotide specialties such as forensic medicine and

triphosphates (dNTPs) producing a comple- tumour pathology. The impact of this develop- J Clin Pathol: first published as 10.1136/jcp.50.10.805 on 1 October 1997. Downloaded from mentary DNA strand. Four different reaction ment on research was acknowledged by mixes, each containing in addition a di- honouring PCR with the title "Major scientific deoxynucleotide triphosphate (ddNTP) corre- development of 1989" and Taq DNA polymer- sponding to one of the four nucleotides, is ase "Molecule of the year 1989" by Science.'9 added to the new DNA strand and terminates Since then, there has been an explosion in the the replication process. This produces DNA number of publications dealing with PCR fragments of various lengths. The sequence is applications. then determined by separating the resulting Owing to financial impact, PCR technology DNA fragments of each reaction by gel has been the subject of ongoing litigation. electrophoresis.' Cetus, the US biotechnology company was For further progress to be made, techniques originally granted the patent to native Taq allowing the production of large quantities of DNA polymerase in 1989. Around the same recombinant DNA were necessary. Cloning time as the 1989 Science article, DuPont was the first revolutionary technique de- challenged the PCR patent. These patents were scribed, involving the isolation and production upheld in February 1991 and by the end of the of many copies of a DNA sequence.'3 14 The year Cetus had disappeared and the Swiss DNA fragment of interest is cut using REs and pharmaceutical company, Hoffmann-La then ligated into other DNA molecules called Roche had acquired the patent rights for native vectors or cloning vehicles (for example, Taq, recombinant Taq polymerase, and the plasmids). The vector and the inserted DNA PCR methodology for US$300 million. The fragment can then be produced in large quan- legal drama continues with Roche now fighting tities by transformed bacteria. Subsequently, the US laboratory supply company, Promega, the cloned sequence can be extracted and ana- over patent rights.20 lysed or used directly as a probe for hybridisation. Cloning is a time consuming process and is Raw materials not routinely applicable to a busy diagnostic Many different types of clinical samples such as pathology laboratory. This obvious disadvan- blood, semen, saliva, single hairs, archival fixed tage was overcome by the development of the paraffin, and plastic embedded tissues can be polymerase chain reaction (PCR). The tech- used for DNA and RNA amplification.2'-26 For nique was first described by Khorana and col- Southern blot DNA detection, large amounts leagues in the early 1970s,'5 but brought to life of DNA are required, which in many cases may and named PCR in 1983 by Kary Mullis, who not be available. Indeed, archival paraffin wax subsequently received the Nobel Prize for embedded material up to 40 years old has been

Chemistry in 1994 for his work on PCR. successfully used for DNA amplification, using http://jcp.bmj.com/ For developing a new method of DNA repli- PCR technology.27 cation, Mullis initially envisaged the hybridisa- Tissues for histopathological examination tion of oligonucleotide primers to single are usually fixed in a suitable fixative to main- stranded DNA and subsequent extension of tain morphology. The commonly used formal- these primers by a DNA polymerase, akin to dehyde fixatives nick DNA and thereby reduce the process of in vivo DNA replication in the maximal size of product that can be ampli-

mammalian cells. After much variation and fied. Good yields of nucleic acid can however on September 25, 2021 by guest. Protected copyright. optimisation of the technique he was able to be obtained using proteolytic enzymes such as amplify a 25 base pair fragment of a plasmid proteinase K or pepsin. using two oligonucleotide primers of 1 1 and 13 The alcohol based fixatives (Carnoy's, bases long.' After this success, the first paper methanol, methanol:acetic acid) also accom- on PCR was published in Science in 1985 by modate PCR amplification to greater or lesser seven scientists (including Mullis) from Cetus, degrees, while mercuric chloride based fixa- a Californian biotechnology company.16 tives largely inhibit PCR.28 The initial PCR method used the Klenow The age of source material for PCR appears fragment of Escherichia coli DNA polymerase I limitless. Even palaeobiological plant matter, to extend the annealed primers. As this enzyme up to 20 million years old has been used.29 is inactivated by the high temperatures used to DNA and RNA can be extracted from speci- melt (denature) DNA strands, it had to be mens by a variety of well described replenished during every cycle after each dena- techniques.30 The precise technique depends turation step. With the discovery of thermosta- on the type and amount of starting material ble DNA polymerases such as Taq (Thermus (fresh or fixed), the amount of potential PCR aquaticus) polymerase, the PCR process be- inhibitors present in the sample, and the type of came simpler, obviating the need for fresh nucleic acid being extracted. PCR inhibitors enzyme addition after each heating step. In are ubiquitous and include potassium ions, addition, these enzymes are active at higher porphyrins from haeme, and other undefined temperature, thus increasing specificity and the products.3' Most extraction methodologies rate of DNA synthesis.'7 18 employ phenol-chloroform extraction, which The subsequent automation of the PCR in most cases eliminates these inhibitors. Many process using dedicated DNA thermal cyclers proprietary DNA and RNA purification kits and its simplicity and ease of use led to its are now available, obviating the need for com- widespread application in disparate scientific plex and time consuming extraction protocols. Origins of .. The polymerase chain reaction in pathology 807

PCR MIX:

i DNA+primers+dNTPs 51____ J Clin Pathol: first published as 10.1136/jcp.50.10.805 on 1 October 1997. Downloaded from + DNA polymerase

5' 3' 3' 5'

Denature Anneal ______31 Extend 3' Parent DNA Primer Denature 11NewVY LVlA%DNA /AHHAnngnltid Extend Cycling continued

Figure 1 Schematic representation ofPCR. Double stranded DNA is denatured by heating. Primers anneal to single stranded DNA and are extended by DNA polymerase. The procedure is repeated over multiple cycles with each amplification step resulting in new strands ofDNA, which subsequently act as templates forfurther amplification.

Technique AMPLIFICATION REFRACTORY MUTATION SYSTEM The originally described PCR technique in- Amplification refractory mutation system volved separating double stranded DNA and (ARMS) is a novel system using primers hybridising oligonucleotide primers (usually designed so that the 3' end coincides with a 17-30 nucleotides in length) to the different mutated nucleotide base, facilitating allelic strands flanking the DNA sequence to be discrimination.36 amplified. New DNA template was created by thermocycling the reaction, through denatura- SINGLE STRAND CONFORMATION POLYMORPHISM tion, annealing, and extension phases (fig 1). Single strand conformation polymorphism Various adaptations of PCR have been (SSCP) is another method used for the detec- developed, many of which are now used in tion of single base changes in DNA and RNA, diagnostic pathology laboratories worldwide. and relies on the different mobilities of DNA strands containing single base pair differences, REVERSE TRANSCRIPTASE PCR when run on non-denaturing polyacrylamide is used 37-39 Reverse transcriptase PCR (RT-PCR) gels. http://jcp.bmj.com/ for detection of RNA targets. In this reaction, copy DNA (cDNA) is created using a reverse DIFFERENTIAL DISPLAY transcriptase enzyme (for example, MMVLV Differential display allows the simultaneous RT) and then subsequent amplification of the genetic analysis of changes in gene expression newly created cDNA follows. Originally the in cells and tissues. The technique uses a set of method used a two step procedure: reverse primers, one of which will hybridise to a poly-

transcription and DNA amplification. The adenylated tail present in mRNA (the primer on September 25, 2021 by guest. Protected copyright. development of rTth polymerase, which com- also contains a one or two base anchor), the bines reverse transcriptase and DNA polymer- other primer is short and arbitrary in sequence, ase activity, obviates the need for a two step and anneals in different positions relative to the reaction. This is a major improvement, as it first primer. A combination of nearly 300 minimises handling and lowers possible con- primers is required to ensure that each possible tamination risks.32 33 mRNA is amplified at least once. The mRNA populations defined by these primers are ASYMMETRIC PCR amplified after reverse transcription and re- Asymmetric PCR is a simple and effective solved on a DNA sequencing gel. Fragments method for the production of single stranded that display differential expression between the DNA suitable for direct sequencing. It uses diseased and non-diseased states can easily be unequal molar concentrations ofprimers in the excised from the gel. Subsequent cloning of reaction set up, essentially driving the reaction individual mRNAs is then possible. The to single target strand accumulation. This can technique has been particularly useful for the easily be sequenced directly, without the need detection of differentially expressed genes in for cloning or the establishment of DNA leukaemia, heart disease, and diabetes libraries.34 mellitus.40

INVERSE PCR CDNA SUBTRACTION PCR Inverse PCR allows amplification of DNA out- cDNA subtraction PCR can be applied easily side the boundaries of known sequences. This to cells enriched by flow cytometry. The is important in the study ofviral tumorigenesis, subtraction procedure involves three steps when attempting to identify possible insertion leading to the identification of a collection of sites of viruses in host DNA, and for the full length cDNAs cloned in an expression vec- assessment of clonality in lymphoid tumours.35 tor, suitable for direct functional analysis. In 808 O'Leary, Engels, Dada

the first step, an RT-PCR is performed that tion. This is achieved using specifically de- amplifies cDNA representing all poly mRNA signed equipment (for example, Perkin Elmer

present in two different samples (X and Y). Applied Biosystems 7700 DNA sequence J Clin Pathol: first published as 10.1136/jcp.50.10.805 on 1 October 1997. Downloaded from The PCR produces 3' cDNA stubs of approxi- detector). Alternatively, an end point format mately 200-600 base pairs that can be can be adopted, in this case using a lumines- amplified through multiple rounds of PCR cence spectrometer. while maintaining the gene expression profile present in the starting mRNAs.41 The second COMPARATIVE GENOME HYBRIDISATION step involves the reciprocal removal of com- Comparative genome hybridisation (CGH) is a mon sequences from both samples using a new approach in fluorescence in situ hybridisa- biotin-avidin cDNA subtraction protocol. tion, allowing the comprehensive analysis of Subtraction product X - Y is enriched for chromosomal imbalances in entire genomes. sequences present in X but not in Y and simi- Genomic DNA from cell populations to be larly Y - X is enriched for sequences in Y, not tested is labelled with modified nucleotides found in X. The final step of the reaction (dig 11 dUTP) and used as a probe to normal involves labelling of the subtraction products metaphase chromosomes of the patient. This X - Y and Y - X, which are used to screen probe is called the test probe. As an internal replica filters from a full length library. cDNA control, genomic DNA derived from cells with clones are selected that hybridise consistently a normal karyotype is differentially labelled with one and not the other subtracted probe. (control DNA probe) and hybridised simulta- neously with the test probe. For detection of REPRESENTATIONAL DIFFERENCE ANALYSIS the hybridised test, and control DNA probes, Representational difference analysis is a new different fluorochromes are used and each is technique that allows the identification of the visualised with epi-fluorescence microscopy differences between two complex genomes.4" with selective filters. Ifthe tissue under analysis The technique basically involves a genomic contains additional chromosomal material, subtractive hybridisation protocol, which al- hybridisation reveals higher signal intensities at lows the investigator to discriminate sequences the corresponding target regions of the hybrid- present in a tumour specimen form normal ised chromosome. Conversely, deletions are control DNA of the same individual. visible as lower signal intensities. By comparing the hybridisation patterns of the test and IN SITU PCR control probes, changes in signal intensities In situ PCR is, for most histopathologists, the caused by allelic imbalance can be conveniently marriage of standard histopathology and mo- identified.45 lecular biology.43 In situ PCR is used to detect single copy target nucleic acid sequences in Applications ofPCR in pathology fixed tissues and cells. It aims to correlate PCR PCR is an established technique and has

results with morphology. While holding the increased the range and sensitivity of diagnos- http://jcp.bmj.com/ greatest potential for diagnostic histopathol- tic procedures. The exquisite sensitivity of ogy, it is a technique that needs to gain PCR is also its major drawback, as contamina- widespread acceptance. tion and amplification artefacts can give rise to difficulties in the interpretation of results. TAQMAN PCR TaqMan PCR (5' nuclease assay) is a major MICROBIOLOGY

advance in PCR. It was first described by Hol- In the past, diagnosis of infections was limited on September 25, 2021 by guest. Protected copyright. land et al,44 who used the 5'-3' endonucleolytic by the supply of appropriate material for Table 1 Microorganisms activity of Taq DNA polymerase to detect tar- culture, protein analysis or microscopy. These that can be detected by PCR get sequences during amplification by PCR. limitations have been overcome by the intro- Viruses Included in the PCR mixture is a probe duction of PCR in diagnostic microbiology. It Adenovirus (usually 20-30 mers in length) designed to is now possible to detect DNA or RNA of Cytomegalovirus hybridise within the target sequence and to be infectious organisms that are either present in Epstein-Barr virus Hepatitis A, B, C non-extendible at the 3' end. The fluorescent small numbers, slow growing (viruses, myco- Herpes simplex virus emission activity of a fluorescent reporter bacteria, etc) or in material not suitable for HIV I and II molecule attached to the probe at its 5' end is culture.46 PCR can facilitate the Human herpesvirus 7, 8 diagnosis of Human papillomavirus neutralised by a quencher molecule at the 3' early and latent stages of infection, which can- HTLV-1 end. When hybridised to its target sequence, not be identified by conventional laboratory Lassa virus the intact shows no of Measles virus probe signal because the techniques. Rotavirus proximity of the reporter molecule to the The examination of archival material allow- quencher molecule. During amplification Taq ing retrospective studies has had great impact Bacteria DNA Mycobacterium tuberculosis polymerase, through its 5'-3' endonu- and has demonstrated correlations between Mycobacterium cleolytic activity, cleaves the probe into frag- viral agents and tumorigenesis (for example, paratuberculosis ments, separating the reporter molecule from human papillomavirus and cervical carcinoma, Mycobacterium leprae the Borrelia burgdoferi quencher, thus allowing its detection. The Epstein-Barr virus and post-transplant lym- Legionella pneumophilia amount of fluorescence is directly proportional phoproliferative disorder (PTLD), and Kaposi Listeria monocytogenes to the amount of specific amplification of the sarcoma herpesvirus/human herpesvirus 8 and Chlamydia trachomatis Helicobacter pylori target. The major advantage of this technique Kaposi's sarcoma). Table 1 lists microorgan- is its ability to detect specifically amplified isms detectable by PCR in routine clinical Protozoa DNA or RNA sequences at selected time samples such as blood, cerebrospinal fluid, Toxoplasma gondii Plasmodium fakiparum points in the PCR, thereby allowing direct semen, saliva, faeces, pleural fluid, and fixed quantitative real time DNA and RNA detec- tissues. Origins of .. The polymerase chain reaction in pathology 809

Table 2 Inherited diseases HUMAN GENETICS Where to from here? that can be screenedfor using a highly specialised research tool with PCR A major use of PCR is in the diagnosis of chro- PCR is mosomal disorders or hereditary diseases, such many uses in medical laboratories. PCR J Clin Pathol: first published as 10.1136/jcp.50.10.805 on 1 October 1997. Downloaded from 3 Thalassaemia as Down's syndrome, thalassaemia, cystic methodology is well established, which greatly a, Antitrypsin deficiency fibrosis, and haemophilia (table 2). Invasive facilitates genetic, microbiological, and viro- Cystic fibrosis The advent of automated ther- Gaucher's disease antenatal procedures to obtain fetal cells, such logical analysis. Haemophilia as chorionic biopsies and amniotic fluid mal cyclers, fluorescent DNA sequencers, and Huntingdon's disease sampling, have an inherent risk to the fetus, real time PCR sequence detectors (ABI 7700 Lesch-Nyhan syndrome sequence the Muscular dystrophy and can perhaps be replaced by non-invasive DNA detector) has also extended Osteogenesis imperfecta techniques. Fetal DNA may be amplified from power and repertoire of PCR. Porphyria The major advance of PCR is that it can Phenylketonuria maternal blood by PCR, and fetal blood cells a sequence of DNA from among the Sickle cell anaemia from maternal blood can be used for aneu- amplify entire genome (three billion Tay Sachs disease ploidy detection and to determine fetal sex.47"" background of the Fetal cells isolated from maternal cervical base pairs in a haploid cell), making it exquisitely more sensitive than other molecular mucus have also been used for genetic analysis.50 Parental testing for genetic disease is biological tools. In the past few years many startling advances made easier by PCR to detect variable have been made in PCR technology including numbers of tandem repeats (VNTRs), micro- in situ PCR, direct sequencing of PCR tandem repeats, and allele specific satellite products, and quantitative assays using ELISA sequences in the parental genome. This can be technology and real time PCR detectors, which achieved using only a few cells with a automatically measure DNA and RNA loads fluorescent multiplex PCR approach, analysing directly in the starting sample. "microsatellite fingerprints" and disease loci in New enzymology including the combined one reaction (B Tutschek, personal communi- reverse transcriptase DNA polymerase enzyme cation). (rTth DNA polymerase), the recently intro- duced long amplifying Taqs (such as TaqXL), BIOLOGY/ONCOLOGY and newer sequencing Taqs (such as Taq CS) TUMOUR will facilitate the investigation ofhuman In oncopathology, PCR has led to a better greatly understanding of the pathobiology of malig- disease, making the basic technique of PCR more robust and more easily reproducible. nancy, allowing the analysis of mutations in Microchip based PCR technologies may oncogenes and tumour suppressor genes (for have once seemed a dream, but we expect they example, c-myc, p53, ras), the detection of will become reality in the next 5-10 years. It is minimal residual disease (MRD), clonality (for that this will utilise a compartmen- in envisaged example, B and T cell gene rearrangements talised solid support with bound reporter in identifying gene rearrange- lymphomas) probes, or acceptor molecules with the poten- ments in follicular lym- (for example, t(14,18) tial to identify point mutations, subchromo- http://jcp.bmj.com/ phomas and the Philadelphia chromosome in somal regions, viruses, bacteria, etc, akin to CML), and in the assessment of loss of hetero- nuclease technology, currently available in zygosity (allelic imbalance) particularly in solution. In cell direct DNA sequencing in time colorectal and breast cancer. PCR's greatest may also be possible if current in situ technolo- versatility is that it allows the examination of gies are refined. formalin fixed paraffin wax embedded tissue, in A simple idea may have far reaching implica-

which DNA may be degraded and is therefore tions; in Kary Mullis's own words, the simple on September 25, 2021 by guest. Protected copyright. not suitable for Southern blotting. Using such method of making unlimited copies of DNA archival material, large scale retrospective although conceived under unlikely circum- genetic analysis of p53, DCC, APC, and ras stances has had far reaching effects.' mutations in colorectal cancer, and genome- wide screening for novel tumour suppressor 1 Mullis KB. The unusual origin of the polymerase chain genes and oncogenes in any cancer can be eas- reaction. SciAm 1990;262:56-61. fluorescent 2 Alberts B, Bray D, Lewis J, Raff M, Roberts K, Watson JD, ily undertaken using PCR and eds. Molecular biology of the cell. 3rd edn. New York: amplicon detection technologies.5l Garland, 1994:291-318. 3 Nathans D, Smith HO. Restriction endonucleases in the analysis and restructuring of DNA molecules. Annu Rev Biochem 1975;44:273-93. FORENSIC PATHOLOGY 4 Southern EM. Detection of specific sequences among DNA PCR has brought significant progress in foren- fragments separated by gel electrophoresis. Mol Biol 1975;98:503-17. sic pathology. It is used in establishing the 5 Warford A. In situ hybridisation: a new tool in pathology. identity of mutilated corpses or decomposed Med Lab Sci 1988;45:381-94. 6 John HA, Birnsteil ML, Jones KW. RNA-DNA hybrids at human remains, in sex determination, in cases the cytological level. Nature 1969;223:582-7. of paternity, and in identifying perpe- 7 Gall JG, Pardue ML. Formation and detection of RNA- disputed DNA hybrid molecules in cytological preparations. Proc trators of crime. This is based on the NatlAcad Sci USA 1969;63:378-83. or restriction 8 Orth G, Jeanteur P, Croissant 0. Evidence for and localisa- amplification of VNTRs frag- tion of vegetative viral DNA replication by autoradio- ment length polymorphisms (RFLPs) and is graphic detection of RNA-DNA hybrids in sections of tumours induced by Shope papilloma virus. Proc Natl Acad referred to as DNA fingerprinting. PCR has Sci USA 1971 ;68:1876-80. not only made such evaluations easier, but also 9 Buongiorno-Nardelli M, Amaldi F. Autoradiographic detection of molecular between rRNA and DNA in tis- even trace amounts or hybrids possible with partially sue sections. Nature 1970;225:946-8. degraded biological material such as blood and 10 Jacob J, Todd K, Birnstiel ML, Bird A. Molecular hybridisation of 'H labelled ribosomal RNA and DNA in ultrathin semen stains or hair, increasing dramatically sections prepared for electron microscopy. Biochim Biophys the range of samples that can be analysed.54 5 Acta 1971;228:761-6. 810 O'Leary, Engels, Dada

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