I Clin Pathol 1997;50:805-810 0

Origins of . .

The polymerase chain reaction in pathology

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

A surprisingly simple method of making unlimitedcopies of DNA fragments conceived under unlikelycircumstances during a moonlit drive through themountains of California.

Kary B Mullis'

The movie J7urassic Park and real life events likethe 0 J Simpson trial have captured publicinterest in molecular biology. The basis ofmolecular biology is the understanding of thestructure and function of DNA, RNA, andproteins, as well as the techniques for manipu-lating these molecules. In this review we dem-onstrate how a single technique revolutionisedthis research area and made molecular biologi-cal methods accessible, not only to researchgroups but also to diagnostic pathology labora-tories. As a result, molecular pathology is nowfirmly established as a pathology discipline,providing new insights into the pathogenesis ofdisease as well as innovative techniques indiagnosis.

Department ofPathology, CornellUniversity MedicalCollege, New York,USAJ J O'Leary

Department ofCellular Science,University of Oxford,Oxford, UKK EngelsM A Dada

Correspondence to:Dr O'Leary, Department ofPathology, Cornell UniversityMedical College, The NewYork Hospital, 1300 YorkAvenue, New York, NY1002 1, USA.

Accepted for publication15 July 1997

Historical backgroundAlthough DNA was first isolated in 1869 byMiescher, its double helix structure was notdescribed until 1953 by Watson and Crick.2 In1955, Arthur Kornberg of Stanford Universitydiscovered DNA polymerase.' This cellularenzyme is involved in DNA replication andrepair by catalysing the addition of nucleotidesto the 3' end of an existing DNA chain. Theinitiation of a new chain requires an existingoligonucleotide or polynucleotide chain, re-

ferred to as a primer. The polymerase attachesnucleotides in a new DNA strand, complemen-tary to nucleotides on corresponding positionsof the parent DNA strand (template strand).RNA polymerases are involved in the assemblyofRNA from a DNA template (transcription).Over time, new tools for producing and

manipulating DNA were developed. Restric-tion endonucleases (RE) cut DNA at specificsequences (restriction sites), making it possibleto isolate strands of DNA containing specificgenes.3 In 1975, Edwin Southern described a

technique for the localisation of specificsequences within genomic DNA by electro-phoretic transfer techniques.4 This technique,subsequently known as Southern blotting,involves the digestion of genomic DNA by one

or more REs and the separation of the resultingfragments by agarose gel electrophoresis. Theseparated fragments of double stranded DNAare then separated into single stranded form

and transferred (blotting) from the gel to asolid support (usually a nitrocellulose or nylonfilter). The sequence of interest can then bedetected using a short fragment of DNA(oligonucleotide probe), which is complemen-tary to the DNA sequence of interest (hybridi-sation).

Initially, radioactive labels (32P, 35S, 3H) wereused for probing, but later non-isotopic labelsincluding biotin, digoxigenin, and fluoresceinwere employed. Three nucleic acid labellingmethods are now described, including enzymeincorporation, chemical derivitisation, andchemical cross linking. The enzymatic labellingreactions for DNA include nick translation,random priming, and 5' and 3' end labellingusing DNA polymerase, Klenow polymerase,and Dnasel.5 For RNA detection, riboprobescan be created using SP6, T3, and T7 in vitroenzymologies. Alternatively, synthetic oligonu-cleotides can be conveniently used as probesfor DNA and RNA detection assays. Addition-ally, PCR probe labelling methods, employingbiotin, digoxigenin or fluorescein labelleddNTPs in substituted molar ratios in the PCRreaction mix, can also be used for generatingprobes for any hybridisation analysis.A refinement of Southern blot analysis, of

interest to pathologists, was first published in1969 by two groups working independently inthe UK and the USA.67 The technique-in situhybridisation-allowed for the first time directcorrelation between hybridisation signals andtissue morphology. The initial reports were fol-lowed by application of the methodology tocryostat, paraffin wax, chromosomal, and elec-tron microscopy preparations.81' Since then,numerous DNA and RNA targets have beendemonstrated using both isotopic and non-isotopic labels.Analogous techniques for RNA and proteins

have been named northern and westernblotting, respectively, as a play on the nameSouthern. These techniques gained wide-spread acceptance in the 1970s.The next significant breakthrough in mo-

lecular biology was the development of rapidDNA sequencing techniques. In the chemicalmethod of Maxem and Gilbert the sequence isdetermined from native DNA itself.'2 DNA islabelled at one end and then exposed to agentsthat destroy one or two of the nucleotidesresulting in fragments that can be separatedand analysed by electrophoresis. The Sanger ordideoxy chain termination method parallels the

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process of DNA replication.2 12 Starting with aprimer, a DNA polymerase adds nucleotidetriphosphates (dNTPs) producing a comple-mentary DNA strand. Four different reactionmixes, each containing in addition a di-deoxynucleotide triphosphate (ddNTP) corre-sponding to one of the four nucleotides, isadded to the new DNA strand and terminatesthe replication process. This produces DNAfragments of various lengths. The sequence isthen determined by separating the resultingDNA fragments of each reaction by gelelectrophoresis.'For further progress to be made, techniques

allowing the production of large quantities ofrecombinant DNA were necessary. Cloningwas the first revolutionary technique de-scribed, involving the isolation and productionof many copies of a DNA sequence.'3 14 TheDNA fragment of interest is cut using REs andthen ligated into other DNA molecules calledvectors or cloning vehicles (for example,plasmids). The vector and the inserted DNAfragment can then be produced in large quan-tities by transformed bacteria. Subsequently,the cloned sequence can be extracted and ana-lysed or used directly as a probe for hybridisa-tion.

Cloning is a time consuming process and isnot routinely applicable to a busy diagnosticpathology laboratory. This obvious disadvan-tage was overcome by the development of thepolymerase chain reaction (PCR). The tech-nique was first described by Khorana and col-leagues in the early 1970s,'5 but brought to lifeand named PCR in 1983 by Kary Mullis, whosubsequently received the Nobel Prize forChemistry in 1994 for his work on PCR.For developing a new method ofDNA repli-

cation, Mullis initially envisaged the hybridisa-tion of oligonucleotide primers to singlestranded DNA and subsequent extension ofthese primers by a DNA polymerase, akin tothe process of in vivo DNA replication inmammalian cells. After much variation andoptimisation of the technique he was able toamplify a 25 base pair fragment of a plasmidusing two oligonucleotide primers of 1 1 and 13bases long.' After this success, the first paperon PCR was published in Science in 1985 byseven scientists (including Mullis) from Cetus,a Californian biotechnology company.16The initial PCR method used the Klenow

fragment of Escherichia coli DNA polymerase Ito extend the annealed primers. As this enzymeis inactivated by the high temperatures used tomelt (denature) DNA strands, it had to bereplenished during every cycle after each dena-turation step. With the discovery of thermosta-ble DNA polymerases such as Taq (Thermusaquaticus) polymerase, the PCR process be-came simpler, obviating the need for freshenzyme addition after each heating step. Inaddition, these enzymes are active at highertemperature, thus increasing specificity and therate ofDNA synthesis.'7 18The subsequent automation of the PCR

process using dedicated DNA thermal cyclersand its simplicity and ease of use led to itswidespread application in disparate scientific

disciplines such as cell biology, and medicalspecialties such as forensic medicine andtumour pathology. The impact of this develop-ment on research was acknowledged byhonouring PCR with the title "Major scientificdevelopment of 1989" and Taq DNA polymer-ase "Molecule of the year 1989" by Science.'9Since then, there has been an explosion in thenumber of publications dealing with PCRapplications.Owing to financial impact, PCR technology

has been the subject of ongoing litigation.Cetus, the US biotechnology company wasoriginally granted the patent to native TaqDNA polymerase in 1989. Around the sametime as the 1989 Science article, DuPontchallenged the PCR patent. These patents wereupheld in February 1991 and by the end of theyear Cetus had disappeared and the Swisspharmaceutical company, Hoffmann-LaRoche had acquired the patent rights for nativeTaq, recombinant Taq polymerase, and thePCR methodology for US$300 million. Thelegal drama continues with Roche now fightingthe US laboratory supply company, Promega,over patent rights.20

Raw materialsMany different types of clinical samples such asblood, semen, saliva, single hairs, archival fixedparaffin, and plastic embedded tissues can beused for DNA and RNA amplification.2'-26 ForSouthern blot DNA detection, large amountsofDNA are required, which in many cases maynot be available. Indeed, archival paraffin waxembedded material up to 40 years old has beensuccessfully used for DNA amplification, usingPCR technology.27

Tissues for histopathological examinationare usually fixed in a suitable fixative to main-tain morphology. The commonly used formal-dehyde fixatives nick DNA and thereby reducethe maximal size of product that can be ampli-fied. Good yields of nucleic acid can howeverbe obtained using proteolytic enzymes such asproteinase K or pepsin.The alcohol based fixatives (Carnoy's,

methanol, methanol:acetic acid) also accom-modate PCR amplification to greater or lesserdegrees, while mercuric chloride based fixa-tives largely inhibit PCR.28The age of source material for PCR appears

limitless. Even palaeobiological plant matter,up to 20 million years old has been used.29DNA and RNA can be extracted from speci-

mens by a variety of well describedtechniques.30 The precise technique dependson the type and amount of starting material(fresh or fixed), the amount of potential PCRinhibitors present in the sample, and the type ofnucleic acid being extracted. PCR inhibitorsare ubiquitous and include potassium ions,porphyrins from haeme, and other undefinedproducts.3' Most extraction methodologiesemploy phenol-chloroform extraction, whichin most cases eliminates these inhibitors. Manyproprietary DNA and RNA purification kitsare now available, obviating the need for com-plex and time consuming extraction protocols.

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Origins of .. The polymerase chain reaction in pathology

PCR MIX:

i DNA+primers+dNTPs 51____+ DNA polymerase

5' 3'

3' 5'

DenatureAnneal _ _____31

Extend 3'Parent DNAPrimer DenatureNew DNA Anngnl

Extend Cyclingcontinued

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

TechniqueThe originally described PCR technique in-volved separating double stranded DNA andhybridising oligonucleotide primers (usually17-30 nucleotides in length) to the differentstrands flanking the DNA sequence to beamplified. New DNA template was created bythermocycling the reaction, through denatura-tion, annealing, and extension phases (fig 1).

Various adaptations of PCR have beendeveloped, many of which are now used indiagnostic pathology laboratories worldwide.

REVERSE TRANSCRIPTASE PCRReverse transcriptase PCR (RT-PCR) is usedfor detection of RNA targets. In this reaction,copy DNA (cDNA) is created using a reversetranscriptase enzyme (for example, MMVLVRT) and then subsequent amplification of thenewly created cDNA follows. Originally themethod used a two step procedure: reversetranscription and DNA amplification. Thedevelopment of rTth polymerase, which com-bines reverse transcriptase and DNA polymer-ase activity, obviates the need for a two stepreaction. This is a major improvement, as itminimises handling and lowers possible con-tamination risks.32 33

ASYMMETRIC PCRAsymmetric PCR is a simple and effectivemethod for the production of single strandedDNA suitable for direct sequencing. It usesunequal molar concentrations of primers in thereaction set up, essentially driving the reactionto single target strand accumulation. This caneasily be sequenced directly, without the needfor cloning or the establishment of DNAlibraries.34

INVERSE PCR

Inverse PCR allows amplification ofDNA out-side the boundaries of known sequences. Thisis important in the study ofviral tumorigenesis,when attempting to identify possible insertionsites of viruses in host DNA, and for theassessment of clonality in lymphoid tumours.35

AMPLIFICATION REFRACTORY MUTATION SYSTEM

Amplification refractory mutation system(ARMS) is a novel system using primersdesigned so that the 3' end coincides with amutated nucleotide base, facilitating allelicdiscrimination.36

SINGLE STRAND CONFORMATION POLYMORPHISM

Single strand conformation polymorphism(SSCP) is another method used for the detec-tion of single base changes in DNA and RNA,and relies on the different mobilities of DNAstrands containing single base pair differences,when run on non-denaturing polyacrylamidegels. 37-39

DIFFERENTIAL DISPLAYDifferential display allows the simultaneousgenetic analysis of changes in gene expressionin cells and tissues. The technique uses a set ofprimers, one of which will hybridise to a poly-adenylated tail present in mRNA (the primeralso contains a one or two base anchor), theother primer is short and arbitrary in sequence,and anneals in different positions relative to thefirst primer. A combination of nearly 300primers is required to ensure that each possiblemRNA is amplified at least once. The mRNApopulations defined by these primers areamplified after reverse transcription and re-solved on a DNA sequencing gel. Fragmentsthat display differential expression between thediseased and non-diseased states can easily beexcised from the gel. Subsequent cloning ofindividual mRNAs is then possible. Thetechnique has been particularly useful for thedetection of differentially expressed genes inleukaemia, heart disease, and diabetesmellitus.40

CDNA SUBTRACTION PCRcDNA subtraction PCR can be applied easilyto cells enriched by flow cytometry. Thesubtraction procedure involves three stepsleading to the identification of a collection offull length cDNAs cloned in an expression vec-tor, suitable for direct functional analysis. In

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the first step, an RT-PCR is performed thatamplifies cDNA representing all poly mRNApresent in two different samples (X and Y).The PCR produces 3' cDNA stubs of approxi-mately 200-600 base pairs that can beamplified through multiple rounds of PCRwhile maintaining the gene expression profilepresent in the starting mRNAs.41 The secondstep involves the reciprocal removal of com-mon sequences from both samples using abiotin-avidin cDNA subtraction protocol.Subtraction product X - Y is enriched forsequences present in X but not in Y and simi-larly Y - X is enriched for sequences in Y, notfound in X. The final step of the reactioninvolves labelling of the subtraction productsX - Y and Y - X, which are used to screenreplica filters from a full length library. cDNAclones are selected that hybridise consistentlywith one and not the other subtracted probe.

REPRESENTATIONAL DIFFERENCE ANALYSISRepresentational difference analysis is a newtechnique that allows the identification of thedifferences between two complex genomes.4"The technique basically involves a genomicsubtractive hybridisation protocol, which al-lows the investigator to discriminate sequencespresent in a tumour specimen form normalcontrol DNA of the same individual.

IN SITU PCRIn situ PCR is, for most histopathologists, themarriage of standard histopathology and mo-lecular biology.43 In situ PCR is used to detectsingle copy target nucleic acid sequences infixed tissues and cells. It aims to correlate PCRresults with morphology. While holding thegreatest potential for diagnostic histopathol-ogy, it is a technique that needs to gainwidespread acceptance.

Table 1 Microorganismsthat can be detected by PCR

VirusesAdenovirusCytomegalovirusEpstein-Barr virusHepatitis A, B, CHerpes simplex virusHIV I and IIHuman herpesvirus 7, 8Human papillomavirusHTLV-1Lassa virusMeasles virusRotavirus

BacteriaMycobacterium tuberculosisMycobacterium

paratuberculosisMycobacterium lepraeBorrelia burgdoferiLegionella pneumophiliaListeria monocytogenesChlamydia trachomatisHelicobacter pylori

ProtozoaToxoplasma gondiiPlasmodium fakiparum

TAQMAN PCRTaqMan PCR (5' nuclease assay) is a majoradvance in PCR. It was first described by Hol-land et al,44 who used the 5'-3' endonucleolyticactivity of Taq DNA polymerase to detect tar-get sequences during amplification by PCR.Included in the PCR mixture is a probe(usually 20-30 mers in length) designed tohybridise within the target sequence and to benon-extendible at the 3' end. The fluorescentemission activity of a fluorescent reportermolecule attached to the probe at its 5' end isneutralised by a quencher molecule at the 3'end. When hybridised to its target sequence,the intact probe shows no signal because of theproximity of the reporter molecule to thequencher molecule. During amplification TaqDNA polymerase, through its 5'-3' endonu-cleolytic activity, cleaves the probe into frag-ments, separating the reporter molecule fromthe quencher, thus allowing its detection. Theamount of fluorescence is directly proportionalto the amount of specific amplification of thetarget. The major advantage of this techniqueis its ability to detect specifically amplifiedDNA or RNA sequences at selected timepoints in the PCR, thereby allowing directquantitative real time DNA and RNA detec-

tion. This is achieved using specifically de-signed equipment (for example, Perkin ElmerApplied Biosystems 7700 DNA sequencedetector). Alternatively, an end point formatcan be adopted, in this case using a lumines-cence spectrometer.

COMPARATIVE GENOME HYBRIDISATIONComparative genome hybridisation (CGH) is anew approach in fluorescence in situ hybridisa-tion, allowing the comprehensive analysis ofchromosomal imbalances in entire genomes.Genomic DNA from cell populations to betested is labelled with modified nucleotides(dig 11 dUTP) and used as a probe to normalmetaphase chromosomes of the patient. Thisprobe is called the test probe. As an internalcontrol, genomic DNA derived from cells witha normal karyotype is differentially labelled(control DNA probe) and hybridised simulta-neously with the test probe. For detection ofthe hybridised test, and control DNA probes,different fluorochromes are used and each isvisualised with epi-fluorescence microscopywith selective filters. If the tissue under analysiscontains additional chromosomal material,hybridisation reveals higher signal intensities atthe corresponding target regions of the hybrid-ised chromosome. Conversely, deletions arevisible as lower signal intensities. By comparingthe hybridisation patterns of the test andcontrol probes, changes in signal intensitiescaused by allelic imbalance can be convenientlyidentified.45

Applications ofPCR in pathologyPCR is an established technique and hasincreased the range and sensitivity of diagnos-tic procedures. The exquisite sensitivity ofPCR is also its major drawback, as contamina-tion and amplification artefacts can give rise todifficulties in the interpretation of results.

MICROBIOLOGYIn the past, diagnosis of infections was limitedby the supply of appropriate material forculture, protein analysis or microscopy. Theselimitations have been overcome by the intro-duction of PCR in diagnostic microbiology. Itis now possible to detect DNA or RNA ofinfectious organisms that are either present insmall numbers, slow growing (viruses, myco-bacteria, etc) or in material not suitable forculture.46 PCR can facilitate the diagnosis ofearly and latent stages of infection, which can-not be identified by conventional laboratorytechniques.The examination of archival material allow-

ing retrospective studies has had great impactand has demonstrated correlations betweenviral agents and tumorigenesis (for example,human papillomavirus and cervical carcinoma,Epstein-Barr virus and post-transplant lym-phoproliferative disorder (PTLD), and Kaposisarcoma herpesvirus/human herpesvirus 8 andKaposi's sarcoma). Table 1 lists microorgan-isms detectable by PCR in routine clinicalsamples such as blood, cerebrospinal fluid,semen, saliva, faeces, pleural fluid, and fixedtissues.

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HUMAN GENETICS

A major use of PCR is in the diagnosis of chro-mosomal disorders or hereditary diseases, suchas Down's syndrome, thalassaemia, cysticfibrosis, and haemophilia (table 2). Invasiveantenatal procedures to obtain fetal cells, suchas chorionic biopsies and amniotic fluidsampling, have an inherent risk to the fetus,and can perhaps be replaced by non-invasivetechniques. Fetal DNA may be amplified frommaternal blood by PCR, and fetal blood cellsfrom maternal blood can be used for aneu-

ploidy detection and to determine fetal sex.47""Fetal cells isolated from maternal cervicalmucus have also been used for geneticanalysis.50 Parental testing for genetic disease ismade easier by PCR to detect variablenumbers of tandem repeats (VNTRs), micro-satellite tandem repeats, and allele specificsequences in the parental genome. This can beachieved using only a few cells with a

fluorescent multiplex PCR approach, analysing"microsatellite fingerprints" and disease loci inone reaction (B Tutschek, personal communi-cation).

TUMOUR BIOLOGY/ONCOLOGYIn oncopathology, PCR has led to a betterunderstanding of the pathobiology of malig-nancy, allowing the analysis of mutations inoncogenes and tumour suppressor genes (forexample, c-myc, p53, ras), the detection ofminimal residual disease (MRD), clonality (forexample, B and T cell gene rearrangements inlymphomas) in identifying gene rearrange-

ments (for example, t(14,18) in follicular lym-phomas and the Philadelphia chromosome inCML), and in the assessment of loss of hetero-zygosity (allelic imbalance) particularly incolorectal and breast cancer. PCR's greatestversatility is that it allows the examination offormalin fixed paraffin wax embedded tissue, inwhich DNA may be degraded and is thereforenot suitable for Southern blotting. Using sucharchival material, large scale retrospectivegenetic analysis of p53, DCC, APC, and ras

mutations in colorectal cancer, and genome-wide screening for novel tumour suppressor

genes and oncogenes in any cancer can be eas-

ily undertaken using PCR and fluorescentamplicon detection technologies.5 l

FORENSIC PATHOLOGY

PCR has brought significant progress in foren-sic pathology. It is used in establishing theidentity of mutilated corpses or decomposedhuman remains, in sex determination, in cases

of disputed paternity, and in identifying perpe-trators of crime. This is based on theamplification of VNTRs or restriction frag-ment length polymorphisms (RFLPs) and is

referred to as DNA fingerprinting. PCR hasnot only made such evaluations easier, but alsopossible even with trace amounts or partiallydegraded biological material such as blood andsemen stains or hair, increasing dramaticallythe range of samples that can be analysed.54 5

Where to from here?PCR is a highly specialised research tool withmany uses in medical laboratories. PCRmethodology is well established, which greatlyfacilitates genetic, microbiological, and viro-logical analysis. The advent of automated ther-mal cyclers, fluorescent DNA sequencers, andreal time PCR sequence detectors (ABI 7700DNA sequence detector) has also extended thepower and repertoire of PCR.The major advance of PCR is that it can

amplify a sequence of DNA from among thebackground of the entire genome (three billionbase pairs in a haploid cell), making itexquisitely more sensitive than other molecularbiological tools.

In the past few years many startling advanceshave been made in PCR technology includingin situ PCR, direct sequencing of PCRproducts, and quantitative assays using ELISAtechnology and real time PCR detectors, whichautomatically measure DNA and RNA loadsdirectly in the starting sample.New enzymology including the combined

reverse transcriptase DNA polymerase enzyme

(rTth DNA polymerase), the recently intro-duced long amplifying Taqs (such as TaqXL),and newer sequencing Taqs (such as Taq CS)will greatly facilitate the investigation ofhumandisease, making the basic technique of PCRmore robust and more easily reproducible.

Microchip based PCR technologies may

have once seemed a dream, but we expect theywill become reality in the next 5-10 years. It isenvisaged that this will utilise a compartmen-talised solid support with bound reporterprobes, or acceptor molecules with the poten-tial to identify point mutations, subchromo-somal regions, viruses, bacteria, etc, akin tonuclease technology, currently available insolution. In cell direct DNA sequencing in timemay also be possible if current in situ technolo-gies are refined.A simple idea may have far reaching implica-

tions; in Kary Mullis's own words, the simplemethod of making unlimited copies of DNAalthough conceived under unlikely circum-stances has had far reaching effects.'

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Table 2 Inherited diseasesthat can be screenedfor usingPCR

3 Thalassaemiaa, Antitrypsin deficiencyCystic fibrosisGaucher's diseaseHaemophiliaHuntingdon's diseaseLesch-Nyhan syndromeMuscular dystrophyOsteogenesis imperfectaPorphyriaPhenylketonuriaSickle cell anaemiaTay Sachs disease

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