Starting a PCR Reaction

A typical PCR reaction set up protocol is shown in Table 6.3. The first step in PCR involves the selection of oligonucleotide primer sequences that are optimal for the amplification of a particular target DNA sequence. In addi- 

Gold (ABI-Perkin Elmer, Foster City, CA). Too much DNA can lead to PCR artifacts and hence should be avoided. The four deoxynucleotide triphosphates (dNTP) include dATP, dCTP, dGTP, and dTTP. The mixture can be prepared and stored in aliquots at — 80°C. The commercial vendors that sell the Taq DNA polymerase enzyme also provide PCR reaction buffer either with or without Mg2+. The basic constituents of PCR buffer include 100 mM Tris-Cl, pH 8.3 (at room temperature), 500 mM KC1, and other additives in some brands. 

Primers can be custom synthesized commercially at a fairly reasonable rate. Primer sequences are selected so that they are typically 21 to 24 nucleotides in length with an average G + C content of 40 to 60%. The primers should not be part of repetitive-sequence DNA, nor should the DNA have palindromic sequences. There are computer programs to help select oligonucleotide primers for PCR, such as PRIMER (from White Head Institute www-genome. wi. mit. echi/ftp //distribution/software/primer. 0.5/manual. asc). In addition, commercial sources exist for software (National Biosciences) that includes the OLIGO program. 

Various thermostable DNA polymerases are available commercially from various vendors. The first thermostable DNA polymerase enzyme that became available commercially was Taq DNA polymerase, isolated from T. aquaticus. This enzyme lacks 3 -to-5 proofreading exonuclease activity and hence has a higher error rate than those enzymes that possess this proofreading activity, such as pfu enzyme. For most routine purposes any thermostable DNA polymerase should suffice, irrespective of its error rate during PCR. 

PCR reaction cycles are carried out in a commercially available programmable thermocycler. A typical PCR cycle consists of initial denatura-tion of DNA at 94°C for 3 to 10 cycles (depending on the enzyme used), followed by 30 to 40 cycles of brief denaturation (94°C, 30 seconds), annealing (50 to 55°C), and elongation (72°C). Variations of these conditions occur if amplifying larger DNA (several kilobase pairs) segments, 

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