Amplification of deoxyribonucleic acid (DNA) fragment using two-step polymerase chain reaction (PCR)

Abstract

Polymerase chain reaction (PCR), an essential tool in many fields such as molecular biology, normally comprises three steps: denaturation at a high temperature, annealing at a low temperature and elongation at a moderate temperature. Here, we report a two-step PCR method which incorporates annealing and elongation step for significant time-saving and reduction in reagent use. To investigate whether the two-step method is as useful as the common three-step method, different lengths of DNA fragments were amplified by two-step PCR and three-step PCR and the influence of incorporated temperature and the amount of Taq polymerases were performed in the present study. The results showed that, all the DNA fragments of 300 to 1000 bp could be amplified by two-step PCR method and the temperature from 50 to 60.8°C could provide a viable range for annealing/extension steps and the fidelity of the PCR has not been affected by the two step method presented. Taken together, the twostep PCR could be used to amplify DNA fragment, which is time-saving, reliable and inexpensive.

Key words: Polymerase chain reaction (PCR), two-step polymerase chain reaction, three-step polymerase chain reaction, time-saving.