Dna

Region of interest

^Primer

Primer

The third and last step of the PCR cycle is the primer extension step (Fig. 7-4). This is essentially when DNA synthesis occurs. In this step, the polymerase synthesizes a copy of the template DNA by adding nucleotides to the hybridized primers. DNA polymerase catalyzes the formation of the phosphodiester bond between an incoming dNTP determined by hydrogen bonding to the template (A:T or G:C) and the base at the 3' end of the primer. In this way, DNA polymerase replicates the template DNA by simultaneously extending the primers on both strands of the template. This step occurs at the optimal temperature of the enzyme, 68°-72°C. In some cases, the annealing temperature is close enough to the extension temperature that the reaction can proceed with only two temperature changes. This is two-step PCR, as opposed to three-step PCR that requires a different temperature for all three steps.

At the end of the three steps, or one cycle (denatura-tion, primer annealing, and primer extension), one copy of double-stranded DNA has been replicated into two copies. Increasing the temperature back up to the denaturing temperature starts another cycle (Fig. 7-5), with the end result being a doubling in the number of double-stranded DNA molecules again (Fig. 7-6). At the end of

■ Figure 7-3 In the second step of the PCR cycle, annealing, the primers hybridize to their complementary sequences on each strand of the denatured template. The primers are designed to hybridize to the sequences flanking the region of interest.

the PCR program, millions of copies of the original region defined by the primer sequences will have been generated (Fig. 7-7). Following is a more detailed discussion of each of the components of PCR.

Components of PCR

The PCR is a method of in vitro DNA synthesis. Therefore, to perform PCR, all of the components necessary for the replication of DNA in vivo are combined in optimal concentrations for replication of DNA to occur in vitro. This includes the template to be copied, primers to prime synthesis of the template, nucleotides, polymerase enzyme, and buffer components including monovalent and divalent cations to provide optimal conditions for accurate and efficient replication.

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