PCR Cloning of Coat Protein Genes

Abstract

The polymerase chain reaction (PCR) is used to amplify DNA between two oligonucleotide primers, of which one is complimentary to a sequence on the (+)-strand and the other to a downstream sequence on the (−)-strand. Amplification between the two primers is achieved by reiterative cycles of template denaturation, primer annealing, and primer extension by a heat-stable DNA polymerase enzyme, which is able to withstand the repeated high temperatures required for DNA denaturation. The products of each reaction cycle serve as templates for subsequent cycles, and so, theoretically, the amount of product doubles after each cycle. PCR is therefore a very sensitive technique that can be used to generate microgram quantities of DNA from as little as a single DNA molecule (see also Chapter 48).