PCR Cloning Using T-Vectors

The polymerase chain reaction (PCR) has revolutionized the way that molecular biologists approach the manipulation of nucleic acids through its ability to amplify specific DNA sequences (1 –3 ). This is achieved by repeated rounds of three different steps: heat denaturation of template DNA, annealing of two convergent oligonucleotide primers to the opposite strands of the DNA template, and then 5′ to 3′ extension from each of the annealed primers using a thermostable DNA polymerase, usually that of Thermus aquaticus (Taq polymerase). Since the product from one round acts as a template for the next, each cycle results in the doubling of target DNA so that the desired sequence accumulates exponentially.