PCR

The goal of PCR, or Polymerase Chain Reaction, is to amplify a specific genetic sequence. In this example, a gene of interest is amplified from purified template DNA.

To perform the amplification, a thermostable DNA polymerase such as Taq polymerase is required to synthesize new DNA strands. It remains functional during the repeated high-temperature steps of PCR.

Primers, which are short single-stranded DNA sequences complementary to regions flanking the gene of interest, are also required. In addition, deoxynucleoside triphosphates, or dNTPs, act as the building blocks of DNA synthesis.

The first step in PCR is heating the reaction mixture to denature the double-stranded DNA into single strands.

Next, the reaction is cooled to allow the primers to anneal to their complementary target sequences. The reaction is then heated to the extension temperature that is optimal for DNA polymerase function.

The polymerase recognizes the primer-template complex and extends the primers by incorporating dNTPs to synthesize new DNA strands.

The reaction then undergoes repeated cycles of denaturation, primer annealing, and extension.

Following the third cycle, eight copies of the target sequence are present. After the fourth cycle, 16 copies are generated. After the fifth cycle, 32 copies are present.

With each cycle, the amount of DNA increases exponentially. After approximately 30 cycles, more than one billion copies of the target sequence can be produced under ideal reaction conditions.