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PCR
Basic Principles and Routine Practice
Lori A. Kolmodin and J. Fenton Williams1. Introduction1.7. PC!? DefinitionThe polymerase chain reaction (PCR) is a primer-mediated enzymatic ampli-fication of specifically cloned or genomtc DNA sequences (I). This PCR pro-cess, nvented by Kary Mullis over 10 years ago, has been automated for routineuse n laboratories worldwide. The template DNA contains the target sequence,which may be tens or tens of thousands of nucleotides in length. A ther-mostable DNA polymerase,
Taq
DNA polymerase, catalyzes the bufferedreaction in which an excess of an oligonucleotide primer pair and four deoxy-nucleoside triphosphates (dNTPs) are used to make millions of copies of thetarget sequence. Although the purpose of the PCR process is to amplifytemplate DNA, a reverse transcription step allows the starting point to beRNA (2-5).1.2. Scope of PCR ApplicationsPCR is widely used in molecular biology and genetic disease research toidentify new genes. Viral targets, such as HIV-l and HCV can be identifiedand quantitated by PCR. Active gene products can be accurately quantitatedusing RNA-PCR. In such fields as anthropology and evolution, sequences ofdegraded ancient DNAs can be tracked after PCR amplification. With itsexquisite sensitivity and high selectivity, PCR has been used for wartimehuman identification and validated in crime labs for mixed-sample forensiccasework. In the realm of plant and animal breeding, PCR techniques are usedto screen for traits and to evaluate living four-cell embryos. Environmental and
From Methods m Molecular Biology, Vol 67 PCR Clonmg Protocols From Molecular Cloningto GenetIc Engmeermg Edlted by* B A White Humana Press Inc , Totowa, NJ
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4 Kolmodin and Williamsfood pathogens can be quickly identified and quantitated at high sensitivity mcomplex matrices with simple sample preparation techniques,1.3. PCR ProcessThe PCR process requires a repetitive series of the three fundamental stepsthat defines one PCR cycle: double-stranded DNA template denaturation,annealing of two oligonucleotide primers to the single-stranded template, andenzymatic extension of the primers to produce copies that can serve as tem-plates in subsequent cycles. The target copies are double-stranded and boundedby annealing sites of the incorporated primers. The 3’ end of the primer shouldcomplement the target exactly, but the 5’ end can actually be a noncomple-mentary tail with restriction enzyme and promoter sites that will also be incor-porated. As the cycles proceed, both the original template and the amplifiedtargets serve as substrates for the denaturation, primer annealing, and primerextension processes, Since every cycle theoretically doubles the amount of tar-get copies, a geometric amplification occurs. Given an efficiency factor foreach cycle, the amount of amplified target, Y, produced from an input copynumber, X; after y1 ycles is
Y
= Xi
1
+ efficiency)(1)With this amplification power, 25 cycles could produce 33 million copies.Every extra 10 cycles produces 1024 more copies. Unfortunately, the processbecomes self-limiting and amplification factors are generally between 105- andlog-fold. Excess primers and dNTPs help drive the reaction that commonlyoccurs n 10 mMTris-HCl buffer (PH 8.3 at room temperature). n addition, 50 mMKC1 is present to provide proper ionic strength and magnesium ion is requiredas an enzyme cofactor (6).The denaturation step occurs rapidly at 94-96°C. Primer annealing dependson the T,, or melting temperature, of the primer:template hybrids. Generally,one uses a predictive software program to compute the T,,,s based on theprimer’s sequence, their matched concentrations, and the overall salt concen-tration. The best annealing temperature is determined by optimization. Exten-sion occurs at 72°C for most templates. PCR can also easily occur with a twotemperature cycle consisting of denaturation and annealing/extension.1.4. Carryover PreventionPCR has the potential sensitivity to amplify single molecules, so PCR prod-ucts that can serve as templates for subsequent reactions must be kept isolatedafter amplification. Even tiny aerosols can contain thousands of copies of car-ried-over target molecules that can convert a true negative into a false positive.In general, dedicated prpeters, plugged pipet tips, and separate work areasshould be designated for pre and post-PCR work. As with any high sensitivity
 
Basic Principles and Routine Practice5technique, the judicious and frequent use of positive and negative controls isrequired (7-9). Through the use of dUTP instead of dTTP for all PCR samples, tis possible to design an internal biochemical mechanism to attack the PCR carry-over problem. PCR products will then be dU-containing and can be cloned,sequenced,and analyzed asusual. Pretreatment of each PCR reaction with uracilN-glycosylase will destroy any PCR product carried over from previous reac-tions, leaving the native T-containing sample ready for amplification (10).1.5. Hot StartPCR is conceptualized as a process that begins when thermal cycling ensues.The annealing temperature sets he specificity of the reaction, assuring that theprimary primer bindmg events are the ones specific for the target in question.In preparing a PCR reaction on ice or at room temperature, however, the reac-tants are all present for nonspecific primer annealing to any single-strandedDNA present. Since Taq DNA polymerase has some residual activity even atlower temperatures, it is possible to extend these misprimed hybrids and beginthe PCR process at the wrong sites! By withholding a key reaction component,such as Tuq DNA polymerase, until an elevated temperature can be reached,the possibility of mispriming is avoided. This can be accomplished by a manualaddition of enzyme above 65-70°C during the first heating ramp to denatur-ation at 94°C. Alternatively, an inactive form of the enzyme AmpliTaq Goldcan be added to all reactions to prevent misprimed extensions. Adding a pre-PCR heat step at 92-95°C for 9-12 min synchronously reactivates the enzymeand achieves an “invisible” hot start. In both cases, the lowest temperatureexperienced by the reaction components is the stringent primer annealing tem-perature, assuring best specificity (II).1.6. PCR AchievementsPCR has been used to speed the gene discovery process and for early detec-tion of viral diseases.Single sperm cells to measure crossover frequencies can beanalyzed and four-cell cow embryos can be typed. Trace forensic evidence ofeven mixed samples can be analyzed. Single copy amplification requires somecare, but is feasible for both DNA and RNA targets.True needlesm haystackscan be found simply by ampli-fling the needles.PCR facilitates cloning of DNA sequenc-ing and forms a natural basis for cycle sequencing by the Sanger method (12).2. PCR Enzymes2.1. AmpliTa@ DNA PolymeraseAmpliTaq DNA Polymerase (Perkin Elmer, Foster City, CA) is a highlycharacterized recombinant enzyme for PCR. It is produced in E. coli from theTaq DNA polymerase gene, thereby assuring high purity. It is commonly sup-plied and used as a 5 U/pL solution in buffered 50% glycerol.
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