Effect of variable annealing temperatures in Polymerase Chain Reaction / Mohd Khairul Shafiq Ali

Polymerase Chain Reaction (PCR) is typically carried om in three distinct steps governed by three distinct temperatures. These three important steps are the denaturing, annealing and extension. Annealing is the second stage of a PCR run whereby the primers will bind to the target DNA or target sequence for amplification. The goal is not to amplify in vitro the entire DNA but only specific region of interest only. In this study, the effects of variable or different annealing temperature on PCR performance were investigated. Six different annealing temperature setting were chosen, that is 46° C, 49°C, ,52°C, 55°C, 58°C, and 61°C. At the end of the PCR run the amplicons were then electrophoresed, stained with ethidium bromide dammed in water and finally photographed by a gel documentation system with UV light. At 46°C to 58°C DNA bands of varying intensity were produced. At 61°C, no band was seen. These results showed that the best annealing temperature (Tm) for this consensus primer was 52°C. The band was clear, sharp and with the highest intensity at the expected 1400 bp molecular weight. This therefore concludes that the Tm of a particular primer sets (forward and reverse) can be experimentally determined to obtain the optimum result.