A Study of PCR Inhibition Mechanisms Using Real Time PCR*^,^†

TLDR

The study investigates how amplicon length, melting temperature, and sequence influence PCR inhibition mechanisms using real‑time PCR. Primers with three amplicon lengths and three melting temperatures were used to target a single allele at the HUMTH01 locus, and varying concentrations of inhibitors were added to measure their effect on amplification efficiency. Different co‑extracted inhibitors produced distinct mechanisms—Taq inhibition, DNA binding, and efficiency loss—sometimes acting in multiple ways, and only some were affected by amplicon size or melting temperature, highlighting the importance of identifying the interfering substance.

Abstract

Abstract: In this project, real time polymerase chain reaction (PCR) was utilized to study the mechanism of PCR inhibition through examination of the effect of amplicon length, melting temperature, and sequence. 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 we find 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.