A Study of PCR Inhibition Mechanisms Using Real Time PCR

Specifically designed primers with three different amplicon lengths and three different melting temperatures were used to target a single homozygous allele in the HUMTH01 locus. The effect on amplification efficiency for each primer pair was determined by adding different concentrations of various PCR inhibitors to the reaction mixture. The results show that a variety of inhibition mechanisms can occur during the PCR process depending on the type of co-extracted inhibitor. These include Taq inhibition, DNA template binding, and effects on reaction efficiency. In addition, some inhibitors appear to affect the reaction in more than one manner. Overall, the project found that amplicon size and melting temperature are important in some inhibition mechanisms and not in others, and the key issue in understanding PCR inhibition is determining the identity of the interfering substance. (publisher abstract modified)