Optimization of Annealing Temperature in Polymerase Chain Reaction on Constructing a Arrestin Variant (Arr3T137W)

Abstract

Polymerase chain reaction (PCR) is one of the most prevalent and effective methods implemented in cloning, sequencing, and DNA profiling. Among the three essential steps in PCR: denaturation (95 °C), annealing and elongation (72 °C), the annealing temperature could impact the amplification of the targeted DNA fragments. This experiment evaluated the effects of annealing temperature on PCR effectiveness and the optimal temperature for maximum yield and precision. To construct a single tryptophan arrestin3 variant, we intend to replace the Threonine with tryptophan by PCR. The effectiveness of this procedure was examined using agarose gel electrophoresis. This enabled comparison to previously effective PCR analysis, for sample identification and product conformation. The control sample at 46°C yielded an accurate and viable PCR product. A deviation of 10°C above or below the control temperature was conducted and analyzed via the same method. The sample at 36°C yielded results similar to the control. However, the sample conducted at 56°C yielded no product due to the annealing efficacy has been dramatically decreased at elevated temperatures. For the tested PCR reaction, annealing temperatures in the range of 36-46°C are effective, whereas temperatures are not effective.