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Polymerase Chain Reaction (PCR) 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;
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