Polymerase Chain Reaction Secondary article (PCR) Article Contents . Polymerase Chain Reaction: Overall Description Michael L Metzker, Baylor College of Medicine, Houston, Texas, USA . DNA Polymerase Reaction Thomas C Caskey, Cogene Biotech Ventures, Houston, Texas, USA . Sensitivity and Contamination of PCR . PCR Introduces Mutations . PCR is a rapid in vitro DNA synthesis process, which can amplify up to a billion copies of a PCR Length Limitations . given nucleic acid target. It has been extensively applied for the identification, detection Creation of Novel Recombinant Molecules by PCR . and diagnosis of genetic and infectious disease. PCR as a Detection System . Degenerate PCR . Ancient DNA Polymerase Chain Reaction: Overall . Quantitative PCR Description . Related Nucleic Acid Amplification Procedures . Ligation Chain Reaction Few techniques rival the impact that the polymerase chain . Summary reaction (PCR) has made in the age of molecular biology. Cloning and deoxyribonucleic acid (DNA) sequencing are other such techniques that have become embedded into synthesis fidelities, respectively. PCR is an elegant but everyday life on the molecular biologist’s bench. Over 60 simple technique for the in vitro amplification of target books alone (not to mention the tens of thousands of DNA utilizing DNA polymerase and two specific oligo- research articles) have been devoted to the strategies, nucleotide or primer sequences flanking the region of methods and applications of PCR for the identification, interest. PCR is a cyclic process of double-strand separa- detection and diagnosis of genetic and infectious diseases. tion of DNA by heat denaturation, specific hybridization Rightfully so, the inventor of PCR, Kary B. Mullis, was or annealing of short oligonucleotide primers to single- awarded the Nobel Prize in Chemistry for his discovery of stranded DNA, and synthesis by DNA polymerase (Saiki the technique in 1993. PCR, however, has not been without et al., 1985; Mullis and Faloona, 1987). Each cycle doubles controversy. In 1989, DuPont challenged the validity of the the region marked by the primer sequences. By sequential Cetus PCR patents in federal court and with the Office of iteration of the process, PCR exponentially generates up to Patents and Trade Marks, and by 1991 the Cetus patents a billion of copies of the target within just a few hours were unanimously upheld and later sold to Hoffman La (Figure 1). Roche for $US300 million. More recently, in 1993, The specificity of PCR is highly dependent on the careful Promega has challenged the validity of the Hoffman La design of unique primers with respect to the genome under Roche Taq DNA polymerase patent which is currently investigation and the nucleotide composition of the primer pending. In this review, we attempt to provide a sequences. Theoretically, a 16-mer (416) is of sufficient comprehensive overview for the molecular biologist when length to represent all unique primer sequences from a applying PCR to his or her application of interest. completely random genome size of 3 billion base pairs. In the real world, however, all genomes are not random and contain varying degrees of repetitive elements. For the human genome, Alus, LINEs (long interspersed DNA DNA Polymerase Reaction elements) and low complexity repeats are frequently observed and should be avoided in primer design when DNA replication is an inherent process for the generation possible. There are a few simple rules for designing primer and evolution of future progeny in all living organisms. At sequences that work well in PCR. In practice, PCR primers the heart of this process is the DNA polymerase which should be between 18 and 25 nucleotides long, have primarily synthesizes new strands of DNA in a 5’!3’ roughly an equal number of the four nucleotides, and show direction from a single-stranded template. Most native a G+C composition of 50–60%. Commercially available DNA polymerases, however, are polyfunctional and show oligonucleotide synthesizers that show phosphamidite 5’-exonuclease and/or 3’-exonuclease activities that are coupling efficiencies 4 98% mean that primers of this size important for cellular DNA repair and proofreading can usually be used in PCR without purification. A variety functions. Numerous molecular biology applications have of computer programs are available for selecting primer harnessed these activities, such as labelling DNA by nick sequences from a target region. Many of these programs translation and TaqMan assays (see below), and end- will reveal internal hairpin structures and self-annealing repair of sheared DNA fragments and improving DNA

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 1 Polymerase Chain Reaction (PCR)

Primer extension

by DNA polymerase Target area 500–3000 bp

Extend

PCR cycle

Separate strands Anneal by heat denaturation primers

Extend Extend

Extend Extend Extend Extend

Extend Extend Extend Extend Extend Extend Extend Extend

25–30 cycles

107–109-fold amplification

Figure 1 The PCR amplification cycle. primer sequences, but manual inspection of the oligonu- 10 mmol L 2 1 tris-HCl, pH 8.3, 50 mmol L 2 1 KCl buffer cleotide is still necessary to maximize successful PCR works well for most PCR applications. The optimal Mg2+ amplifications. concentration, however, may need to be determined The concentrations of the PCR cocktail ingredients are empirically for difficult target templates. The performance also important for product specificity, fidelity and yield. In and fidelity of Taq DNA polymerase are sensitive to the addition to Taq DNA polymerase and primers, the PCR free Mg2 1 concentration (Eckert and Kunkel, 1990), mixture contains the cofactor magnesium ion (Mg2+), the which ionically interacts with not only the dNTPs but also four 2’-deoxyribonucleoside-5’-triphosphates (dNTPs) with the primers, the template DNA, ethylenediaminete- and the buffer. In general, PCR reagent concentrations traacetic acid (EDTA), and other chelating agents. In most that are too high from ‘standard conditions’ result in cases, the Mg2+ concentration will range from 1.0 to nonspecific products with high misincorporation errors, 4.0 mmol L 2 1. and those that are too low result in insufficient product. A The number of cycles and the cycle temperature/length typical 50-ml PCR cocktail that contains 0.4 mmol L 2 1 of of time for template denaturation and primer annealing each primer, 200 mmol L 2 1 of each dNTP, 1.5 mmol L 2 1 and extension are important parameters for high-quality MgCl2, and 1.25 units Taq DNA polymerase in PCR results. The optimal number of cycles is dependent on

2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Polymerase Chain Reaction (PCR) the starting concentration or copy number of the target splashes, the use of positive displacement pipettes, adding DNA and typically ranges from 25 to 35 cycles. Too many DNA last, and carefully choosing positive and negative cycles will significantly increase the amount of nonspecific controls (Kwok and Higuchi, 1989). Contamination is PCR products. For low copy number targets, such as the likely to surface for DNA samples that are difficult to integrated provirus of Human immunodeficiency virus type amplify because of sequence content, or due to poor primer 1 (HIV-1) from human genomic DNA, two rounds of PCR design and chemical impurities in DNA extractions. This is are employed first using an outer primer pair set followed especially true for low copy number targets or degraded by an internal (nested) primer pair set flanking the region of samples, as greater numbers of amplification cycles are interest to yield positive and specific PCR products. Brief generally required to achieve the desired product. In these but effective denaturation conditions, that is 94–978C for cases, residual amounts of exogenous DNAs can compete 15–30 s, are necessary as Taq DNA polymerase has a half- and override the amplification process, resulting in life of only 40 min at 958C. Annealing conditions, on the spurious data. The best approach to challenge dubious other hand, are dependent on the concentration, base results is to repeat the experiment with scrupulous care to composition and the length of the oligonucleotide and details and controls. Biological samples collected at a typically range between 55 and 688C for 30–60 s. The single time point should be divided into multiple aliquots length of the amplified target is directly proportional to the such that independent DNA extractions and PCR experi- primer extension length of time. Primer extension is ments can be performed to verify and validate initial performed between 68 and 728C and, as a rule of thumb, results. Data should be discarded if inconsistent positive is approximately 60 s for every 1 kb. and negative PCR results occur upon repetition of the Crude extracts from blood, cerebral spinal fluid, urine, experiment. While negative controls can rule out reagent buccal smears, bacterial colonies, yeast spores, etc. are contamination, sporadic contamination can go un- routinely used as sources of DNA for PCR templates. Due checked. The probability of repeating spurious contam- to the high sensitivity of PCR, rapid isolation protocols, ination in a consistent manner is extremely low. such as heat and detergent disruptions, and enzymatic There are three sources of contaminating DNA: (1) digestion of biological samples have been frequently used. carryover contamination from previously amplified PCR Caution should be invoked when using crude extracts as products; (2) crosscontamination between multiple source starting materials for PCR amplifications because a materials; and (3) plasmid contamination from a recombi- number of impurities are known to inhibit Taq DNA nant clone that contains the target sequence. Of the three, polymerase. These include red blood cell components, carryover contamination is considered to be the major sodium dodecyl sulfate (SDS), high salts, EDTA and too source of contamination because of the relative abundance much DNA. Since only a few hundred target molecules are of amplified target sequences. The substitution of dUTP needed for successful PCRs, in most cases, these impurities for dTTP in the PCR cocktail has been routinely used as a can be effectively removed by simply diluting the starting method of preventing carryover contamination. Pretreat- material. Each sample should then be tested with control ment of subsequent PCR mixtures prior to thermal cycling primers that specifically amplify a known target to with uracil DNA glycosylase results in the removal of dU determine the integrity of the crude extract. Alternatively, from any carryover PCR product, but does not affect the the isolation of the desired organism, such as HIV-1, template DNA or dUTP. The dU removal creates an human Hepatitis A virus, influenza virus, cytomegalovirus, abasic site that is heat labile and degrades during thermal etc., or the isolation of specific cell fractions, such as cycling, thus preventing carryover amplification. More- peripheral blood mononuclear cells, can significantly over, ultraviolet light can reduce work surface and reagent increase the sensitivity and specificity of the PCR contamination. Cross-contamination between samples is amplifications. more difficult to diagnose, and suspicious results should be repeated from independent DNA extracts and PCR experiments for samples in question. Plasmid contamina- tion, on the other hand, can be identified by sequence Sensitivity and Contamination of PCR analysis and comparison to all laboratory plasmid sequences. Contamination is the dark side of the PCR force. The exquisite sensitivity of PCR can result in contamination from even a single molecule of foreign or exogenous DNA (Gibbs and Chamberlain, 1989; Kwok and Higuchi, 1989). PCR Introduces Mutations To minimize false positives, standard operating procedures have been described, including the physical isolation of The power and ease of PCR, however, were not fully PCR reagents from the preparation of DNA templates and appreciated until the introduction of the thermostable PCR products, using autoclaved solutions, premixing and DNA polymerase isolated from Thermus aquaticus (Taq) aliquoting reagents, the use of disposable gloves, avoiding (Saiki et al., 1988) and automated instrumentation in 1988.

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 3 Polymerase Chain Reaction (PCR)

It was here that PCR could be run in fully closed and PCR Length Limitations automated systems. Fresh Klenow DNA polymerase did not have to be added at each cycle and PCR could be For most applications, standard PCR conditions can performed at higher annealing and extension tempera- reliably amplify target sizes up to 3–4 kb from a variety of tures, which increased the specificity and yields of the source materials. Target sizes greater than 5 kb, however, reactions while minimizing the risks of contamination. A have been described in the literature using standard PCR ‘hot start’ PCR further enhances specificity by preventing conditions, but generally yield low quantities of PCR the formation of nonspecific products that arise during the product. The PCR size limitation can be attributed to the initial steps of thermal cycling in PCR. misincorporation of nucleotides that occurred 1 in 4000– Taq DNA polymerase has been shown to incorporate 5000 bp that ultimately reduced the efficiency of amplifying nucleotides incorrectly at a frequency of 1 in 9000 bases by longer target regions. A breakthrough in long PCR came a mutation reversion assay (Tindall and Kunkel, 1988). through the combined use of two thermostable DNA From sequence analysis of cloned PCR products, a slightly polymerases, one of which contains a 3’-exonuclease higher error frequency was determined (1 in 4000–5000 bp) activity (Barnes, 1994; Cheng et al., 1994). The principle for Taq DNA polymerase (Innis et al., 1988). The fidelity of for long PCR is that the Taq DNA polymerase performs DNA synthesis for Taq DNA polymerase, however, can the high-fidelity DNA synthesis part of PCR, coupled with vary significantly with changes in free Mg2 1 concentra- the proofreading activity of Pfu, Vent or Pwo DNA tion, changes in the pH of the buffer, or an imbalance in the polymerases. Once the nucleotide error is corrected, Taq four dNTP concentrations. Polymerase misincorporation DNA polymerase can then complete the synthesis of long errors are minimized when the four dNTPs are equimolar PCR templates. From empirical studies, only a trace and between 50 and 200 mmol L 2 1 (Innis et al., 1988). Since amount of the 3’-exo DNA polymerase, roughly 1% to that Taq DNA polymerase lacks a 3’-exonuclease activity, of Taq DNA polymerase or another DNA polymerase misincorporated bases typically cause chain termination of isolated from Thermus thermophilis (Tth), is needed to DNA synthesis that are not propagated in subsequent perform long PCRs greater than 20 kb. Other important PCR cycles. In a worst-case scenario, a mutation occurring factors for long PCR are the isolation of high-quality, high- during the first round of PCR from a single target molecule molecular weight DNA and protection against template and propagated thereafter would exist at a frequency of damage, such as depurination during thermal cycling. The 25% in the final PCR product. Since hundreds of target use of the cosolvents glycerol and dimethylsulfoxide copies are routinely used as starting DNA in PCR and (DMSO) have been shown to protect against DNA most misincorporations terminate DNA synthesis, the damage by efficiently lowering the denaturation tempera- observed error frequency is significantly ! 25%. ture by several degrees centigrade. The rule of thumb for Cloning of full-length genes from PCR products, primer extensions still applies for long PCRs (60 s kb 2 1), however, has been problematic because PCR-induced although for targets greater than 20 kb, times extension mutations can cause amino acid substitutions in the wild- should not exceed 22 min per cycle. The complexity and type sequence. Thus, significant effort must be employed in size of the genome under investigation can also affect the the complete sequencing of multiple PCR clones to identify size of long PCR products. For example, PCR product mutation-free clones or ones that contain synonymous lengths of 42 kb have been described for the amplification substitutions that do not change the protein coding of l bacteriophage DNA (Barnes, 1994; Cheng et al., sequence. Accordingly, thermostable DNA polymerases 1994), compared with a 22 kb PCR product obtained from that contain a 3’-exonuclease (3’-exo) activity for proof- the human b-globin gene cluster (Cheng et al., 1994). reading of misincorporated bases have been recently introduced and include DNA polymerases isolated from Pyrococcus furiosus (Pfu), Thermococcus litoralis (Vent), Pyrococcus species GB-D (Deep Vent) and Pyrococcus Creation of Novel Recombinant woesei (Pwo). The error frequencies of these DNA Molecules by PCR polymerases are 2–6-fold less than Taq DNA polymerase (Cline et al., 1996), but these polymerases are difficult for PCR can amplify both single-stranded and double- routine use, as the 3’-exonuclease activity can easily stranded DNA templates as well as complementary degrade the single-stranded PCR primers. 3’-Exo DNA DNA (cDNA) from the reverse transcription of messenger polymerases, however, have been successfully used in long ribonucleic acid (mRNA) templates. Because of the PCR in combination with Taq DNA polymerase and show flexibility of automated DNA synthesis, in vitro mutagen- an approximately twofold lower error frequency than Taq esis experiments can easily be performed by PCR. DNA polymerase alone (Cline et al., 1996). Recombinant PCR products can be created via the primer sequences by tolerated mismatches between the primer and the template DNA or by ‘5’-add-on’ sequences. Primer- mediated mutagenesis can accommodate any nucleotide

4 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Polymerase Chain Reaction (PCR) substitutions and small insertions or deletions of genetic PCR as a Detection System material. The desired genetic alteration can be moved to any position within the target region by use of two PCR is a powerful tool for the detection of human overlapping PCR products with similar mutagenized ends polymorphic variation that has been associated with (Figure 2, left). This is accomplished by denaturing and hereditary diseases. Many PCR techniques have been reannealing the two overlapping PCR products to form described that can discriminate between wild-type and heteroduplexes that have 3’-recessed ends. Following the mutant alleles, but in this section only a few of the most extension of the 3’-recessed ends by Taq DNA polymerase, frequently used techniques are discussed. Of these, DNA the full-length recombinant product is reamplified with the sequencing of PCR products is the most widely used and outer primers only to enrich selectively the full-length most sensitive method for the detection of both novel and recombinant PCR product. 5’-Add-on adapters can also be known polymorphic differences between individuals. used to join two unrelated DNA sequences, such as the Complementing scanning technologies, however, have splicing of an exogenous promoter sequence with a gene of been developed for the rapid detection of allelic differences interest (Figure 2, right). The promoter–gene sequences are because of the high costs associated with DNA sequencing joined at the desired junction by 5’-add-on gene specific and the ability to process large numbers of samples. Single- and 5’-add-on promoter-specific adapters that can PCR strand conformation polymorphism (SSCP) has been amplify the promoter and the gene targets, respectively. commonly used as a technique for the identification of Heteroduplexes can then be formed, as described above, genetic polymorphisms. Following PCR, the product is from the two overlapping PCR products, which are then heat denatured and subjected to native or nondenaturing selectively amplified with outer primers to generate the gel electrophoresis. Allelic differences between samples are desired full-length recombinant PCR product. detected as mobility band shifts by radioactive and

Promoter X Gene X

PCR individually 5’-end gene Y Mismatch in sequence primer sequence to target Remove primers and Promoter Y Gene Y denature/reanneal

5’-end promoter X sequence

Remove primers and denature/reanneal

3’-end DNA synthesis

PCR with outer primers only

Figure 2 Creation of mutagenized or recombinant PCR products via primer mismatches (left) or 5’-add-on sequences (right).

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 5 Polymerase Chain Reaction (PCR) nonradioactive labelling procedures. PCR-SSCP, how- sequence information (Lee et al., 1988). PCR primer ever, is limited in fragment size to about 200 bp because the sequences are derived from the reverse translation of 6–9 accuracy in discriminating between different alleles amino acid codons, which will result in varying levels of diminishes significantly with an increase in the fragment degeneracy except for methionine and tryptophan resi- length. dues. Careful attention should be exercised in the design of Multiplex PCR allows for the simultaneous amplifica- degenerate primers because increasing the primer complex- tion of multiple target regions and has been particularly ity (i.e. using codons that show more than twofold useful for the detection of exon deletion(s) in X-linked degeneracy) will typically result in an increase in non- disorders, such as Duchenne muscular dystrophy (Cham- specific PCR products. One approach in reducing the berlain et al., 1988) and Lesch–Nyhan syndrome (Gibbs complexity of the degenerate primer is the use of codon bias et al., 1990). The multiplex PCR products are resolved by for the particular organism from which the gene will be gel electrophoresis and are visualized by ethidium bromide cloned. Alternatively, the alignment of orthologous gene staining. The absence of specific PCR product(s) is sequences from other species can greatly improve the diagnostic of exon deletion(s) in affected males, and specificity of cloning the gene of interest by revealing ‘half-dosage’ PCR products are diagnostic of carrier evolutionarily conserved domains. Once the optimal mothers (Metzker et al., 1995). Moreover, up to 46 primer primer sequence is determined, the mixture of oligonucleo- pairs have been simultaneously amplified by multiplex tides can be simultaneously synthesized and will represent PCR with excellent success (90%) for the large-scale all possible amino acid combinations of the degenerate identification of human single nucleotide polymorphisms sequence. The specificity of PCR should then selectively (SNPs) by hybridization to high-density DNA chip arrays. amplify the correct primer sequences to generate a gene or Genetic polymorphisms can also be identified by gene family-specific probe from which the full-length immobilizing the PCR product on to a nylon membrane cDNA can be obtained. Degenerate PCR has been in a dot blot format and probing by hybridization with an successfully used in the screening of novel gene family allele-specific oligonucleotide (ASO) that contains a 5’- members such as G-protein-coupled receptors, nuclear biotin group. ASO hybridization is detected by adding steroid receptors and protein tyrosine kinases. streptavidin-horse radish peroxidase, which binds to the biotinylated probe, followed by a colorimeter assay. The colorimeter ASO assay has been applied to the genotyping of human leucocyte antigen (HLA)-DQA alleles and the Ancient DNA detection of b-thalassaemia mutations. More recently, multiplex PCR and colorimeter ASO methodologies have Phylogenetics is the study of evolutionary relationships been combined in a reverse fashion, in which ASOs are between specimens that are inferred from contempora- immobilized on to nylon membrane strips and probed neous sequences. The ability to obtain DNA sequences against biotinylated gene-specific multiplex PCR reac- from specimens or even fossils that are millions of years old tions. Allele-specific PCR products are detected by could equip the phylogeneticist with a powerful means of hybridization and conversion of a colourless substrate to directly testing an a priori hypothesis. Following death of a blue precipitate for the simultaneous genotyping of the tissue or organism, however, DNA is rapidly degraded HLA-DQA1, low-density lipoprotein receptor, glycophor- by, presumably, nuclease activities and hydrolytic pro- in A, haemoglobin G gammaglobin, D7S8, and group- cesses, resulting in short fragment sizes that are generally specific component. no longer than 100–150 bp. Moreover, this ‘old’ DNA is Lastly, in situ PCR enables the amplification of target largely modified by oxidative processes and by intermole- sequences from sections of formalin-fixed, paraffin-em- cular crosslinks that render it unsuitable for cloning by bedded tissue specimens to determine the levels of gene standard molecular biology procedures. Short PCRs, expression in specific cell types that otherwise could not be however, have been successfully performed from DNA detected by conventional in situ hybridization. PCR is samples isolated from archival and ancient specimens performed directly on glass slides by overlaying the PCR (Pa¨ a¨ bo, 1989). mixture on to the specimen, sealing the slides to prevent Museums hold vast collections of archived hospital files evaporation, and temperature cycling using a thermal of patient specimens and of different species that have been sensor or modified thermal cycler that holds glass slides. collected over the last century. In a recent study, phylogenetic analyses of DNA sequences were performed from reverse transcriptase PCR (RT-PCR) of formalin- fixed, paraffin-embedded tissue specimens obtained from Degenerate PCR US servicemen killed in the 1918 ‘Spanish’ influenza pandemic. Viral sequences from three different gene Degenerate PCR is a powerful strategy for obtaining novel regions were consistent with a novel H1N1 Influenza A full-length cDNA sequences from limited amino acid virus that was most closely related to influenza strains that

6 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Polymerase Chain Reaction (PCR) infect humans and swine, but not wild waterfowl, accurately the starting amounts of target sequences. considered to be the natural reservoir for the influenza Unlike gel electrophoresis, real-time QPCR has the unique virus (Taubenberger et al., 1997). Moreover, PCR and advantage of being a closed-tube system, which can DNA sequencing have been performed on DNA extrac- significantly reduce carryover contamination. Using this tions of archaeological findings, such as amplifying technique, one can easily monitor and quantitate the mitochondrial DNA sequences from a 7000-year old accumulation of PCR products during log phase amplifi- human brain, amplifying both mitochondrial and nuclear cation. The TaqMan assay utilizes dual reporter and DNA sequences from bone specimens from a 14 000-year- quencher fluorescent dyes that are attached to a non- old saber-toothed cat, and amplifying chloroplast DNA extendible probe sequence. During the extension phase of sequences from fossil leaf samples from a 17 million-year- PCR, the 5’-nuclease activity of Taq DNA polymerase old Miocene Magnolia species. cleaves the hybridized fluorogenic probe, which releases the reporter signal and is measured during each cycle. In addition to real-time QPCR, TaqMan assays have broad utility for the identification of SNPs. Quantitative PCR

Quantitative PCR (QPCR) has been widely used for detecting and diagnosing genetic deletions, for studying gene expression and for estimating the viral load of HIV-1. Related Nucleic Acid Amplification While DNA quantitation by multiplex PCR has been Procedures previously described (Metzker et al., 1995), the quantita- tion of RNA has been wide reaching for the latter two Other in vitro systems can amplify nucleic acid targets such areas. For many applications, estimating the relative as the transcription-based amplification system (TAS) amount of PCR product is sufficient to describe a (Kwoh et al., 1989), its more recent version called the self- biological observation. The absolute quantitation of sustained sequence replication (3SR) (Guatelli et al., 1990) RNA molecules, however, has been more difficult than and the ligation-dependent Qb-replication assay (Tyagi for DNA because of the difficulty of generating accurate et al., 1996). These methods are best suited for the detection controls. Internal standards derived from synthetic RNA and semiquantitation of RNA target sequences. The or cRNA have been designed to contain the same primer strategy for TAS and 3SR is a continuous series of reverse sequences as the target but yield a different-sized PCR transcription and transcription reactions that mimic product that can be easily separated by gel electrophoresis. retroviral replication by amplifying specific RNA se- cRNAs are not only coamplified with target sequences, but quences via cDNA intermediates. The primers contain 5’- also coreverse transcribed to account for the variable add-on sequences for T7, T3 or SP6 promoters that are efficiencies of cDNA syntheses. Moreover, QPCR is incorporated into the cDNA intermediates. The rapid typically performed in the exponential or log phase of the kinetics of transcription-based amplifications is an attrac- amplification process (typically 14–22 cycles) to obtain tive feature of these systems, which can amplify up to 107 accurate quantitative results. The absolute amount of molecules in 60 min. Short amplified products, however, target mRNA can be quantitated by serial dilutions of the which are due to incomplete transcription of the target target/internal control mixture and by extrapolating region and incomplete RNase H digestion of the RNA– against the standard curve. DNA hybrids, can be problematic in the TAS and 3SR Both the variable range of initial target amounts and the assays. presence of various inhibitors can, however, adversely Unlike PCR, TAS or 3SR assays, the ligation-dependent affect the kinetics and efficiencies of PCR. Alternatively, a Qb-replication assay results in the amplification of probe, strategy based on a quantitative competitive (QC) not target, sequences. This assay utilizes a single hybridiza- approach has been used to minimize the effects of these tion to the target sequence, which is embedded within, and variables. Known quantities of the competitor template, divided between, a pair of adjacently positioned midvar- which contains the same primer sequences as the target but iant (MDV-1) RNA probes. MDV-1 RNA is the naturally differs in size, are introduced into replicate PCRs contain- occurring template for the bacteriophage Qb RNA repli- ing identical quantities of the target. The point at which the case. Following the isolation of the probe–target hybrids, intensities of the PCR products derived from the target ligation of the binary probes creates a full-length amplifi- sequence and the competitor template are equivalent is able MDV-1 RNA reporter. When Qb replicase is added, used to estimate the amount of target sequence in the newly synthesized MDV-1 RNA molecules are amplified original sample (Gilliland et al., 1990). from ligated binary probes that originally hybridized to the Recently, real-time QPCR and QCPCR (Heid et al., target sequence (Tyagi et al., 1996). Similar to TAS and 1996) using a 5’-nuclease fluorogenic or TaqMan assay 3SR, the Qb-replication assay shows rapid kinetics, (Holland et al., 1991) has been developed to measure generating up to 109 molecules in 30 min, and all three

ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net 7 Polymerase Chain Reaction (PCR) methods have been successfully used for the detection and applications for PCR seems endless and it is truly a quantitation of HIV-1 RNA molecules. remarkable technique that has been widely used in molecular biology.

Ligation Chain Reaction References Barany F (1991) Genetic disease detection and DNA amplification using The ligase chain reaction (LCR) can also amplify short cloned thermostable ligase. Proceedings of the National Academy of DNA regions of interest by iterative cycles of denaturation Sciences of the USA 88: 189–193. and annealing/ligation steps (Barany, 1991). LCR utilizes Barnes WM (1994) PCR amplification of up to 35-kb DNA with high four primers: two adjacent ones that specifically hybridize fidelity and high yield from l bacteriophage templates. Proceedings of to one strand of target DNA and a complementary set of the National Academy of Sciences of the USA 91: 2216–2220. adjacent primers that hybridize to the opposite strand. Chamberlain JS, Gibbs RA, Rainier JE, Nguyen PN and Caskey CT LCR primers must contain a 5’-end phosphate group, such (1988) Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification. Nucleic Acids Research 23: 11141– that thermostable ligase (Barany, 1991) can join the 3’-end 11156. hydroxyl group of the upstream primer to the 5’-end Cheng S, Fockler C, Barnes WM and Higuchi R (1994) Effective phosphate group of the downstream primer. Successful amplification of long targets from cloned inserts and human genomic ligations of adjacent primers can subsequently act as the DNA. Proceedings of the National Academy of Sciences of the USA 91: LCR template, resulting in an exponential amplification of 5695–5699. the target region. LCR is well suited for the detection of Cline J, Braman JC and Hogrefe HH (1996) PCR fidelity of Pfu DNA SNPs because a single-nucleotide mismatch at the 3’ end of polymerase and other thermostable DNA polymerases. Nucleic Acids Research 24: 3546–3551. the upstream primer will not ligate and amplify, thus Eckert KA and Kunkel TA (1990) High fidelity DNA synthesis by the discriminating it from the correct base. Although LCR is Thermus aquaticus DNA polymerase. Nucleic Acids Research 18: generally not quantitative, linear amplifications using one 3739–3744. set of adjacent primers, called the ligase detection reaction, Gibbs RA and Chamberlain JS (1989) The polymerase chain reaction: a can be quantitative. Coupled to PCR, linear ligation assays meeting report. Genes and Development 3: 1095–1098. can also be used as a mutation detection system for the Gibbs RA, Nguyen PN, Edwards A, Civitello AB and Caskey CT (1990) identification of SNPs using both wild-type-specific and Multiplex DNA deletion detection and exon sequencing of the hypoxanthine phosphoribosyltransferase gene in Lesch–Nyhan fa- mutant-specific primers in separate reactions. The oligo- milies. Genomics 7: 235–244. nucleotide ligase assay was first reported to detect SNPs Gilliland G, Perrin S, Blanchard K and Bunn HF (1990) Analysis of from both cloned and clinical materials using a 5’-end cytokine mRNA and DNA: detection and quantitation by competitive biotin group attached to the upstream primer and a polymerase chain reaction. Proceedings of the National Academy of nonisotopic label attached to the downstream primer Sciences of the USA 87: 2725–2729. (Landegren et al., 1988). Allele-specific hybridizations and Guatelli JC, Whitfield KM, Kwok DY et al. (1990) Isothermal, in vitro ligations can be separated by immobilization to a amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proceedings of the National Academy of Sciences streptavidin-coated solid support and directly imaged of the USA 87: 1874–1878. under appropriate conditions without the need for gel Heid CA, Stevens J, Livak KJ and Williams PM (1996) Real time electrophoretic analysis. quantitative PCR. Genome Research 6: 986–994. Holland PM, Abramson RD, Watson R and Gelfand DH (1991) Detection of specific polymerase chain reaction product by utilizing the 5’!3’ exonuclease activity of Thermus aquaticus DNA polymerase. Summary Proceedings of the National Academy of Sciences of the USA 88: 7276– 7280. Some of the general concepts and practices of PCR have Innis MA, Myambo KB, Gelfand DH and Brow MAD (1988) DNA been reviewed here. Not only has PCR made a major and sequencing with Thermus aquaticus DNA polymerase and direct significant impact on basic and clinical research but it has sequencing of polymerase chain reaction-amplified. Proceedings of the National Academy of Sciences of the USA 85: 9436–9440. also been well accepted and utilized in forensic science. For Kwoh DY, Davis GR, Whitfield KM et al. (1989) Transcription-based any scientific methodology to be accepted in the courts as amplification system and detection of amplified human immunodefi- evidence, it must satisfy four criteria: that the method (1) be ciency virus type 1 with a bead-based sandwich hybridization format. subject to empirical testing, (2) be subject to peer review Proceedings of the National Academy of Sciences of the USA 86: 1173– and publication, (3) has a known error rate, and (4) is 1177. generally accepted in the scientific community. The Kwok S and Higuchi R (1989) Avoiding false positives with PCR. Nature application of PCR has been admitted in the US courts 339: 237–238. Landegren U, Kaiser R, Sanders J and Hood L (1988) A ligase-mediated as evidence in criminal cases for the analysis of human gene detection technique. Science 241: 1077–1080. DNA sequences, and in January 1997 as evidence for the Lee CC, Wu X, Gibbs RA et al. (1988) Generation of cDNA probes phylogenetic analysis of HIV DNA sequences (State of directed by amino acid sequence: cloning of urate oxidase. Science 239: Louisiana v. Richard J Schmidt, 1997). Clearly, the scope of 1288–1291.

8 ENCYCLOPEDIA OF LIFE SCIENCES / & 2001 Nature Publishing Group / www.els.net Polymerase Chain Reaction (PCR)

Metzker ML, Allain KM and Gibbs RA (1995) Accurate determination Taubenberger JK, Reid AH, Krafft AE, Bijwaard KE and Fanning TG of DNA in agarose gels using the novel algorithm GelScann(1.0). (1997) Initial genetic characterization of the 1918 ‘Spanish’ influenza Computer Applications in the Biosciences 11: 187–195. virus. Science 275: 1793–1796. Mullis KB and Faloona FA (1987) Specific synthesis of DNA in vitro via Tindall KR and Kunkel TA (1988) Fidelity of DNA synthesis by the a polymerase-catalysed chain reaction. Methods in Enzymology 155: Thermus aquaticus DNA polymerase. Biochemistry 27: 6008–6013. 335–351. Tyagi S, Landegren U, Tazi M, Lizardi PM and Kramer FR (1996) Pa¨ a¨ bo S (1989) Ancient DNA: extraction, characterization, molecular Extremely sensitive, background-free gene detection using binary cloning, and enzymatic amplification. Proceedings of the National probes and Qb-replicase. Proceedings of the National Academy of Academy of Sciences of the USA 86: 1939–1943. Sciences of the USA 93: 5395–5400. Saiki RK, Scharf S, Faloona F et al. (1985) Enzymatic amplification of b- globin genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 230: 1350–1354. Further Reading Saiki RK, Gelfand DH, Stoffel S et al. (1988) Primer-directed enzymatic Erlich HA, Gelfand D and Sninsky JJ (1991) Recent advances in the amplification of DNA with a thermostable DNA polymerase. Science polymerase chain reaction. Science 252: 1643–1650. 239: 487–491. Innis MA, Gelfand DH, Sninsky JJ and White TJ (eds) (1990) PCR State of Louisiana v. Richard J Schmidt (1997) Reasons for ruling of Protocols: A Guide to Methods and Applications. San Diego: Academic Louisiana State 15th Judicial District Court Judge Durwood Conque. Press. 15th Judicial District Court, Lafayette Parish, Louisiana. Criminal Mullis KB, Ferre´F and Gibbs RA (eds) (1994) The Polymerase Chain Docket 73313. Reaction. Boston: Birkha¨ user.

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