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Current Diagnostic Pathology (2003) 9, 159--164 c 2003 Published by Elsevier Science Ltd. doi:10.1016/S0968 - 6053(02)00102-3 MINI-SYMPOSIUM: ADVANCES IN LABORATORY PRACTICE Polymerase chain reaction and its applications N. Bermingham and K. Luettichw w Department of Pathology, Education and Research Building, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland; and Department of Dermatology, Weill Medical College of Cornell University,1300 York Avenue, New York, NY10021, USA KEYWORDS PCR; RT-PCR; primer design; gene expression; applications Summary The polymerase chain reaction and its expanding numbers of modif|cations have become a mainstayin diagnostic and research medicine.The technique allows amplif|cation of nucleic acid sequences both for the purposes of disease and pathogen detection and also for the preparation of hybridization probes and sequencing templates. PCR mimics the in vivo process of DNA replication with a sensitivity which enables detection of a single target sequence in 106 genomes. The principles of standard PCR, together with its more widely-used variations, are reviewed and their main applications outlined. c 2003 Published by Elsevier Science Ltd. Since its development in the 1960s and 1970s,1 amplif|cation of nucleic acid sequences has revolutionized much of diagnostic and research molecular science. The polymerase chain reaction (PCR) is now a basic part of a large proportion of assay protocols. In this article, the principles of PCR are reviewed and the main PCR variations and applications are outlined. PCR has become a mainstay of the use of molecular science in diagnosis and research. Central to molecular diagnostics and assays are the nucleic acids -- the building blocks of nature. Nucleic acids [deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)] form the basis for storage and processing of the genetic information; abnormalities of their structure and function are responsible for the majority of disease, both inherited and acquired, and the detection of such alterations is the aim of a large proportion of laboratory molecular medicine. STRUCTURE OF DNA AND RNA DNA is a stable, double-stranded polymeric molecule that exists mainly as a right-handed double helix.The basic component is the nucleotide, which comprises one of four paired deoxyribonucleotides, each consisting of a sugar molecule, a base and a triphosphate group. The backbone of DNA is the deoxyribose sugar connected Correspondence to: NB.Tel.: +3531 809 3701; Fax: +3531 837 0687; E-mail: [email protected] by phosphate groups. Phosphodiester bonds between the 30 -carbon atom of one sugar and the 50 -carbon atom of the next give the backbone its structure and its 50 to 30 direction. By convention, the sequence of nucleotide bases is written in this direction. Linked to the 10-carbon atom of each sugar is one of four possible bases. The bases of DNA and RNA are heterocyclic aromatic rings with a variety of substituents. Adenine and guanine are purines (bicyclic) while cytosine, thymine and uracil are monocyclic pyrimidines. Structurally, DNA is described best using the Watson--Crick model of base pairing; two opposing DNA strands (image/mirror image) wind around a common axis forming a double helix, thus positioning bases to the inside with phosphate groups and sugar moeities turned outwards. Both strands are connected to each other via hydrogen bonds formed between base pairs where adenine always interacts with thymine (or uracil) and cytosine pairs with guanine. The sequence of one chain determines the other, making the two chains complementary. RNA differs from DNA in that the sugar molecule is ribose rather than deoxyribose, and the base uracil is substituted for thymine. DNA, which contains the genetic information, is transcribed to information-transmitting molecules of RNA [messenger RNA (mRNA)].These mRNA molecules subsequently serve as a template for gene products where protein is synthesized (translation). A sequence of three nucleotide base pairs represents a code for an amino acid (codon), and this codon sequence determines a sequence of amino acids (a protein). 160 PCR PCR is the in vitro enzymatic synthesis and amplif|cation of specif|c DNA sequences.2 PCR technology began with the discovery of the f|rst DNA polymerase around 1955. The enzyme was purif|ed in 1958, but automation and modern PCR technology was not developed until 1983. The discovery of thermostable polymerase enzymes revolutionized PCR making automation and rapid reactions possible. PCR mimics the in vivo process of DNA replication. It begins with one molecule of the double-stranded DNA to be amplif|ed, called the target or template DNA. Following separation (denaturation) of the target sequence, a pair of short synthetic DNA sequences called oligonucleotides or primers is bound to the template (annealing/ hybridization).These serve as a starting point for the addition of nucleotides by DNA polymerases. Here, the original template determines the sequence of nucleotides added. In vitro, this process can be achieved by cyclical alterations of temperature facilitating the DNAstrand separation, hybridization of primers and polymerization as follows. First, target DNA is separated into two strands by heating to 92--981C. The temperature is then reduced to between 37 and 551C to allow the primers to anneal (the actual temperature depends on the primer lengths and sequences). Following annealing, the temperature is then increased to 60 --721C for optimal polymerization. In the f|rst polymerization step, the target is copied from the primer sites for various distances on each target molecule until the beginning of Cycle 2, when the reaction is heated to 951C again which denatures the newly synthesized molecules. In the second annealing step, the primer can bind to the newly synthesized strand, and during polymerization can only copy until it reaches the end of the f|rst primer. Thus at the end of Cycle 2, some newly synthesized molecules of the correct length exist. In subsequent cycles, these soon outnumber the target molecules and increase two-fold with each cycle. If PCR was 100% eff|cient, one target molecule would become 2n after n cycles.3 In practice, 20 -- 40 cycles are commonly used. If a pair of oligonucleotide primers can be designed to be complementary to the target molecule such that they can be extended by a DNA polymerase towards each other, then that target region can be greatly amplif|ed. Due to the extreme amplif|cation achievable, it has been demonstrated that PCR can sometimes amplify as little as one molecule of the starting template;4 PCR can also detect one copy of DNA in 106 genomes. Thus, any source of DNA that provides one or more target molecules can, in principle, be used as a template for PCR. This includes DNA prepared from blood, tissue, forensic specimens, palaeontological samples or in the laboratory from bacterial colonies or phage plaques. Whatever the CURRENT DIAGNOSTIC PATHOLOGY source, PCR can only be applied if some sequence information is known so that primers can be designed.5 Primers In most applications, it is the sequence and combination of the primers that determine the overall assay success. Therefore, primer design should follow certain rules:6 primers should lie within highly conserved regions of the genome of interest; they should be 14 -- 40 nucleotides long with a balanced G+C content of around 50%; they must not be complementary to each other (to prevent primer dimer formation) and should lack secondary structure. Primers are designed to anneal to opposite strands of the target sequence so that they will be extended towards each other by addition of nucleotides to their 30 ends. Short target sequences amplify more easily so often this distance is o500 base pairs, but with optimization, PCR can amplify fragments of 410kbp in length. If the sequence to be amplif|ed is known, the primer design is relatively easy owing to the multitude of computational tools for primer design available today. If it is not known, then primer design is more diff|cult but may still be possible by using degenerate primers. DNA polymerases The f|rst enzyme to be used was the large fragment of DNA polymerase I from Escherichiacoli (the Klenow fragment) for primer extension at 371C. This enzyme had a number of drawbacks as it is not stable at high temperatures and therefore required repeated addition after the denaturation step of each cycle. In addition, target DNAs of greater than a few hundred base pairs could not be amplif|ed. A major advance came with the discovery of the thermostable Taq DNA polymerase, f|rst found in Thermus aquaticus.7 Optimal PCR extension occurs at 721C withTaq polymerase in the presence of magnesium ions and dNTPs, and the enzyme remains active at this temperature even after repeated exposures to 941C during the denaturation step. This polymerase is more specif|c, produces less background in PCR than the Klenow fragment, and can amplify targets up to 10 kb. A variety of DNA polymerases have also been characterized from other Thermus species such asTth (Thermus thermophilus) and Tfl (Thermus flavus). Cyclers As mentioned before, microprocessor-controlled machines called thermal cyclers/thermocyclers allowed for the automation of PCR and are now the most convenient method for performing PCR in the laboratory. Modern cyclers feature user-def|ned, temperature-controlled PCR AND ITS APPLICATIONS sample blocks or wells providing both uniform temperature adjustment during cycling and high cycling time reproducibility. PCR reactions are not usually 100% eff|cient and the reaction conditions need to be varied to improve eff|ciency. This is very important when trying to amplify a particular target from a population of other sequences, e.g. one gene from genomic DNA. The usual parameters to vary include the annealing temperature and the magnesium concentration. TYPES OF PCR Standard PCR This entails the amplif|cation of a specif|c sequence, as outlined above, and is used routinely to prepare more DNA targets for subsequent analyses. Multiplex PCR 161 ing fragments are ligated under conditions that favour intramolecular circulation of individual fragments. If a linearized vector DNA of known sequence is present at this stage, the DNA fragments will be incorporated into the vector and can then be analysed by targeted amplif|cation with primers complementary to fragment-flanking vector sequences. Differential PCR This entails the simultaneous amplif|cation of a gene of interest and a control gene. Both genes are amplif|ed in the sample reaction tube and, following electrophoresis, the products are then analysed using a densitometer where the intensity of the gene of interest is compared with the intensity of the control gene. Originally, this technique was used to detect gene copy number changes, for example c-erbB2 in cancer samples, but has found a more widespread application for semiquantitative genetic assays.10 In situ PCR This involves the addition of a number of different primer pairs to the same reaction mixture for the detection of multiple abnormalities or infectious agents in the same assay. Multiplex PCR is particularly helpful to detect mutations, deletions, insertions and re-arrangements in clinical specimens. This is a method which combines PCR amplif|cation of target DNA/RNA with localization of these sequences in intact tissue or cytological specimens. This technique entails amplif|cation taking place followed by direct visualization of positive areas/cells using in situ hybridization. Reverse transcriptase-PCR TaqMan assays This begins with the conversion of RNA to complementary DNA (cDNA) by a reverse transcriptase enzyme. This is an extremely sensitive means of analysing gene expression.8 TaqMan assays exploit the fact that Taq polymerase possesses a 50 --30 exonuclease activity in addition to its 50 -30 -polymerase activity.TaqMan PCR differs from conventional PCR by the addition of a third oligonucleotide, called the TaqMan probe, to the PCR reaction. The TaqMan probe is a dually labelled oligonucleotide complementary to the PCR product to be amplif|ed. A fluorescent dye, called reporter, is covalently linked to the 50 end of the probe whereas another dye, the quencher, is linked to its 30 end. As long as the probe is intact, fluorescence emission is negligible due to Forstertype energy transfer. During PCR cycling, both primers and probe anneal to their target sequences. While elongating the primers, Taq polymerase will reach the 50 end of the probe and hydrolyse it thus releasing the reporter from the influence of the quencher. An increase in fluorescence can be measured, and fluorescence data can then be analysed qualitatively and quantitatively. Nested PCR This is a form of re-amplif|cation of a PCR product. In other words, two separate amplif|cations are used to improve the outcome of a PCR. The f|rst PCR uses a set of primers that yields a large product which is then used as a template for the second amplif|cation. The second set of primers anneals to sequences within the initial product producing a second smaller product. Nested PCR increases the specif|city of the reaction since formation of the f|nal product depends upon the bonding of two separate sets of primers and because two sets of amplif|cation (each of the order of 25 cycles) are used.9 Inverse PCR This allows the amplif|cation of DNA sequences that lie outside of known sequence boundaries.Genomic DNA is f|rst digested into small fragments with a restriction enzyme that cleaves genomic DNA frequently. The result- APPLICATIONS OF PCR There are two main types of PCR application: 1. Analytical PCR -- often from unique samples, e.g. detection of infectious agents in patient samples, 162 genetic analysis, tumour diagnostics, research and forensics. 2. Preparative PCR, e.g. synthesis of hybridization probes and sequencing templates (Tables1 and 2). PCR analysis of gene mutations allows both nature and location of the mutation to be determined. It is ideally suited to providing numerous DNA templates for mutation screening. Partial DNA sequences at the genomic or cDNA level from a gene associated with disease enable gene-specif|c PCR reactions to be designed. Amplif|cation of the appropriate gene segment then allows rapid testing for the presence of associated mutations in large numbers of individuals.11 Restriction fragment length polymorphisms Restriction fragment length polymorphisms (RFLPs) designate genetic variations in alleles characterized by the presence or absence of a specif|c restriction site. Such polymorphisms can be typed using Southern-blot hybridization. A DNA probe representing the locus is hybridized against genomic DNA samples that have been digested with the relevant restriction enzyme and size fractionated using agarose gel electrophoresis. The resulting restriction fragments represent the two alleles corresponding to the absence or presence of the restriction site. PCR can provide an alternative to this method; primers can be designed to flank the polymorphic restriction site and used for amplif|cation of genomic DNA. The resulting PCR products can then be digested with the appropriate restriction enzyme and analysed by gel electrophoresis. RFLP is a technique useful in the determination of clonality, loss of heterozygosity (LOH) and, more recently, methylation. CURRENT DIAGNOSTIC PATHOLOGY A wide variety of PCR-based methods can be used to test for known mutations. Small insertions or deletions can be detected by designing primers complementary to regions flanking the mutation site, and distinguishing the normal and mutant alleles by gel electrophoresis. If the mutation changes a restriction site, then mutant and normal alleles can be distinguished by amplifying across the mutant site and digesting the PCR product with the relevant restriction endonuclease. Even if the mutation does not result in a restriction site difference, it may be possible to exploit the difference between wild-type and mutant by amplif|cationcreated restriction site PCR. Oligonucleotide primers can be designed to discriminate between target DNA sequences that differ by a single nucleotide in the region of interest. Genomic DNA can be amplif|ed using a primer that creates a specif|c restriction site within either one of the alleles that is not present in the other allele. Sequence variation can then be analysed using restriction digestion.This method can be used to type specif|c alleles at a polymorphic locus and is called allele-specif|c PCR. In another form of allele-specif|c PCR, a primer is designed to have a mismatched nucleotide at the extreme 30 end. Since specif|c and eff|cient priming of DNA synthesis is crucially dependent on correct base pairing at the 30 end, mispaired sequences are less readily amplif|ed. As a result, one of the alleles can be selectively amplif|ed for further analysis. This method has found particular use in detecting a specif|c pathogenic mutation and is also called amplif|cation refractory mutation system (ARMS) test.12 Short tandem repeats Short tandem repeats (STRs, microsatellites) comprise a def|ned region of DNA containing multiple copies of Table 1 Applications of PCR 1. Identif|cation and analysis of mutations in eukaryotic DNA -- DNA deletions and insertions can be detected by a change in the size of the PCR product. -- Failure to produce any PCR fragment indicates thatthe primer is found in an area of deletion. -- Mutations can be identif|ed by hybridizing PCR-generated DNA fragments to radioactively labelled RNA probes and digesting the DNA--RNA complexes with RNase A.Digestion will occur if the complex contains any mismatches. -- Localization of mutations has also been achieved by examining PCR products using denaturing gradient gel electrophoresis (DGGE).This separates about 50% of DNA strands o1000 bp in length that have single base changes. 2. Detection of amplif|ed oncogenes -- differential PCR --The oncogene of interest and a single-copy reference gene are co-amplif|ed in the same PCR tube.Gene amplif|cation is determined by comparing the intensity of the oncogene PCR product with that of the amplif|ed reference single-copy gene. 3. Detection of pathogenic organisms in clinical samples 4. Identif|cation of biological and forensic samples 5. Gene polymorphisms 6. Gene expression -- Growth factors during wound healing. -- Gene expression during embryogenesis. -- Quantitation of gene expression in various organs, e.g. dystrophin. PCR AND ITS APPLICATIONS Table 2 Summary of general applications of PCR Typing genetic markers -- RFLPs, STRPs DNAtemplates for mutation screening Detection of point mutations cDNA cloning Genomic DNA cloning Genome walking DNA sequencing Gene expression studies In vitro mutagenesis 163 naturation, annealing and DNA synthesis, as in PCR.The difference from conventional PCR is that only one primer is used and ddNTPs are present in the reaction mixture. The product will accumulate linearly rather than exponentially due to the presence of only one primer. Fluorescent labelling systems with the use of different fluorophores in the four base-specif|c reactions, and automated detection technology allows for accurate automated DNA sequencing.13 Mutagenesis short sequences of bases which are repeated a number of times. The number of repeats, typically between one and f|ve nucleotides long, varies among individuals in the population. STRs can be typed conveniently by PCR. Primers are designed from sequences known to flank a specif|c STR locus, permitting amplif|cation of alleles whose sizes differ by integral repeat units. The PCR products can then be size fractionated by gel electrophoresis. Cloning The major advantages of PCR as a cloning method are its rapidity, sensitivity and robustness. DNA cloning by PCR can be performed in a matter of hours, compared with cell-based cloning which may take weeks. Cell-based cloning of PCR amplif|cation products, however, is often required to permit subsequent structural and functional studies.Various plasmid-cloning systems are used to propagate PCR-cloned DNA in bacterial cells; once cloned, the insert can be cut out and analysed further. DNA sequencing DNA sequencing usually involves enzymatic DNA synthesis in the presence of base-specif|c dideoxynucleotide (ddNTPs) chain terminators. The DNA to be sequenced is used in a single-standed form, and DNA polymerase synthesizes new complementary DNA strands.The reactions are carried out using one or more labelled nucleotides and a sequencing primer. In addition, the mixture contains the base-specif|c ddNTPs, which differ from normal dNTPs in that they lack a hydroxyl group at the 30 -carbon position as well as at the 20 -carbon position. When incorporated into a growing DNA chain, the ddNTP cannot participate further in phosphodiester bonding at its 30 carbon and therefore causes termination of chain synthesis beyond that point in the chain. Double-stranded DNA templates can be used in standard sequencing by denaturing the DNA prior to oligonucleotide binding. However, the quality of sequences produced may be poor. In cycle sequencing (linear amplif|cation sequencing), a thermostable DNA polymerase is used together with a temperature cycling format of de- Mutagenesis is a technique of changing the base sequence of DNA in order to test its effect on gene or DNA function. Mutagenesis can be carried out in vitro or in vivo. In vitro, a gene may be cloned and single-stranded recombinant DNA recovered. A mutagenic oligonucleotide primer is then designed which has, at the intended mutation site, a single base difference, coding for the intended mutation.This is then allowed to prime new DNA synthesis to create a complementary full-length sequence containing the desired mutation. The new DNA is then used to transform cells and to study the effects of the mutation. Genetic testing In terms of genetic testing, information can be acquired in two ways: direct testing, where a sample is tested to see whether a certain genotype is present in that individual; and gene tracking, where linked markers are used in family studies. Direct testing is the optimal means of laboratory diagnosis, and its role expands as more and more specif|c genetic abnormalities are identif|ed. The gene and abnormality of interest must be known, as well as the relevant normal (wild-type) sequence. Direct testing is almost always done by PCR, and can be applied to a wide range of tissue samples including blood, buccal scrapes, chorionic villous samples, hair or semen etc. in forensic samples, archived pathological specimens and Guthrie cards. Regardless of the application, of paramount importance in all types of PCR is rigorous laboratory technique to prevent contamination and false-positive results. As PCR is so sensitive, contamination with even small amounts of exogenous material can result in its amplif|cation. A number of precautions can be taken to minimize this risk including separate rooms for preparation and post-PCR analysis, the wearing of protective clothing and gloves, and ultraviolet-mediated DNA crosslinking. From nucleic acid extraction to performance of the amplif|cation steps, care must be taken to preserve sample integrity, and the inclusion of positive and negative controls in each assay is crucial to result validation. 164 CURRENT DIAGNOSTIC PATHOLOGY DNA-based diagnosis, therefore, relies heavily on PCR, both for geneticists, research scientists and microbiologists/virologists. Detection of infectious agents can be performed using PCR techniques for an increasingly large number of pathogens, both for diagnostic and prognostic purposes (e.g. human papillomavirus testing in cervical intra-epithelial neoplasia). PCR-based techniques are also central to the screening and diagnosis of genetic disorders such as cystic f|brosis, and for the diagnosis of cancer and determination of recurrences and prognostic factors. PCR generation of sequences also forms the basis for microarray technology (gene chips), a dynamic and rapidly expanding area in research. The role of PCR remains central in the setting of increasing importance of molecular techniques in diagnostic pathology. PRACTICE POINTS * * * * * PCR forms the basis of much of diagnostic and research medicine Amplif|cation of both DNA and RNA (RT-PCR) sequences can be performed allowing examination of gene expression A large range of sample types may be examined,including forensic or paleontological specimens Various modif|cations of PCR have allowed for vast expansion of the diagnostic and research uses The development of fluorogenic probes and realtime quantitative PCR allows greater automation of the technique without the requirement of subsequent detection steps REFERENCES 1. Templeton N S. The polymerase chain reaction--history, methods and applications. Diagn Mol Pathol 1992; 1: 58--72. 2. Erlich H A. PCR Technology: Principles and Applications for DNA Amplification. New York: Stockton, 1989. 3. Mullis K B. The unusual origin of the polymerase chain reaction. Sci Am 1990; 4: 56--65. 4. Li H, Gyllenstein U B, Cui X, Saiki R K, Ehrlich H, Arnheim N. Amplification and analysis of DNA sequences in single human sperm and diploid cells. Nature 1988; 335: 414--417. 5. Turner P C, McLennan A G, Bates A D, White M R H. Instant Notes in Molecular Biology. BIOS Scientific Publications, 1997. 6. Taylor G R. Polymerase chain reaction: basic principles and automation. In: McPherson M J, Quirke P, Taylor G R (eds). PCR: a Practical Approach. Oxford: IRL Press, 1992. 7. Chien A, Edgar D B, Trela J M. Deoxyribonucleic acid polymerase from the extreme thermophile Thermusaquaticus. J Bacteriol 1976; 127: 1550--1557. 8. Baumforth K R N, Nelson P N, Digby J E, O’Neill J P, Murray P G. The polymerase chain reaction. J Clin Pathol Mol Pathol 1999; 52: 1--10. 9. Kawasaki E S. Amplification of RNA. In: Innis M A, Gelfand D H, Stunski J J (eds). PCR Protocols, a Guide to Methods and Applications. New York: Academic Press, 1990; 21--27. 10. Reimer T, Luettich K, Gerber B. Epidermal growth factor receptor gene and c-erbB-2 gene amplification in ovarian cyst fluid. Obstet Gynaecol 1996; 88: 967--972. 11. Stachan T, Read A P. Human Molecular Genetics. Bios Scientific Publishers Ltd, 1999. 12. Newton C R, Graham A, Heptinstall L E et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res 1989; 17: 2503--2516. 13. Wilson R K, Chen C, Ardalovic N, Burns J, Hood L. Development of an automated proceedure for fluorescent DNA sequencing. Genomics 1990; 6: 626--634.