Biotechnology and Bioinformatics (1+1)

4.1.2. Steps involved in PCR

4.1.2.1. DNA denaturation

Denaturation occurs when the reaction is heated to 92-96 ° C. The time required to denature the DNA depends on its complexity, the geometry of the PCR tube, the thermal cycler, and the volume of the reaction. For DNA sequence s with high G+C content larger denaturation time is required. Template DNA strands are entirely separated. Gene rally, 94-96 ° C for 2-3 minutes is sufficient.

4.1.2.2. Annealing

The oligonucleotide primer s hybridize to their complementary single - stranded target sequence s. The temperature of this step varies from 37 to 65 ° C, depending on the homology of the primers for the target sequence as well as the base composition of the oligo nucleotide s. Primers are present at a significantly greater concentration than the target DNA, and are shorter in length. As a rule, lowering the annealing temperature from the calculated Tm will increase the likelihood of non-specific amplification. As the temperature is increased through Tm, specificity will increase and yield will decrease.

4.1.2.3. Extension

Last step is the extension of the oligonucleotide primer by a thermostable polymerase . Temperature of 72 ° C is used for extension. The time required to copy the template fully depends on the length of the PCR product. The extension rates of thermostable polymerases are between 2 and 4 kbp min. Significant breakthrough in PCR is that it can amplify segments of up to 45 kb efficiently.