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 hybridisa- tion. 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