Polymerase Chain Reaction

In a typical PCR, successive synthetic cycles are performed in which DNA polymerase copies a target DNA sequence from a template molecule in vitro (Fig. 1). The amplification products of each cycle provide new templates for the next round of amplification. Thus, the concentration of the target DNA sequence increases exponentially over the course of PCR. The typical PCR mixture contains (1) a thermostable DNA polymerase (Taq poly-merase), (2) target-specific forward and reverse oligodeoxynu-cleotide primers, (3) each of the four deoxynucleotide triphosphates (dNTPs), (4) reaction buffer, and (5) a source of template (genomic DNA, cDNA, or a cell lysates). The target sequence is defined by the specificity of the oligodeoxynu-cleotide primers that anneal to complementary sequences on opposite template strands flanking the region of interest. During the PCR, these primers are extended in the 5'^3' direction by the polymerase enzyme to yield overlapping copies of the original template. Each cycle of PCR proceeds through three distinct phases: (1) denaturation, (2) primer annealing, and (3) primer extension. The denaturation step is typically accomplished by incubation of samples for 1 min at 94oC to unwind the DNA containing the gene of interest. The primer annealing step is accomplished at a temperature that is specific for the PCR primers and conditions employed, typically 1 min at 55-65oC.

Microsatellite Instability Pcr

Fig. 1. Polymerase chain reaction. In each cycle of PCR, sequence-specific primers flanking a target sequence anneal to template DNA, and primer extension is accomplished using Taq polymerase (or other thermostable polymerase enzyme). In the first cycle of PCR, the template is composed entirely of input sample (genomic DNA or cDNA). With the second and subsequent cycles of PCR, newly synthesized PCR products become templates for additional amplification. The amplified products accumulate exponentially during a typical 25- to 35-cycle PCR.

Fig. 1. Polymerase chain reaction. In each cycle of PCR, sequence-specific primers flanking a target sequence anneal to template DNA, and primer extension is accomplished using Taq polymerase (or other thermostable polymerase enzyme). In the first cycle of PCR, the template is composed entirely of input sample (genomic DNA or cDNA). With the second and subsequent cycles of PCR, newly synthesized PCR products become templates for additional amplification. The amplified products accumulate exponentially during a typical 25- to 35-cycle PCR.

During the annealing step, oligodeoxynucleotide primers recognize and hybridize to the target sequence contained within the single-stranded template. The primer extension step is accomplished at 72oC (for Taq polymerase) for 1-2 min. During this step, the polymerase enzyme catalyzes the polymerization of dNTPs in a DNA-directed DNA synthesis reaction. The actual times used for each cycle will vary (from 15 s to 1-2 min) depending on the type of thermocycler employed and its temperature ramping speed. Amplification of target sequence is accomplished through repetition of these incubations for 25-30 (or more) cycles. The exact number of cycles necessary to produce an amplicon that can be detected (visualized or otherwise detected) will depend on the starting concentration of the target sequence. By the end of the third cycle of amplification, a new double-stranded molecule is formed in which both the 5' end and 3' end coincide exactly with the primers employed (Fig. 1). Because the copy number theoretically doubles after each successive cycle, an exponential increase of 2n (n is the total number of cycles) is accomplished during the complete chain reaction. Accumulation of amplicons corresponding to the target sequence eventually reaches a plateau. The initial number of target sequences contained within the template sample, the efficiency of primer extension, and the number of PCR cycles performed determine the upper limits of amplification. The majority of double-stranded products formed during subsequent cycles of PCR are of a defined size and are seen clearly as a sharp band when analyzed by gel electrophoresis.

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