PCR Inhibitory Factors: Final Report.

Date: 31/03/2000	Ref No: LGC/VAM/2000/027	Type: Report
Author(s): Dawson C
Organisation/Publisher: LGC

This report outlines the basic kinetic theory of the Polymerase Chain Reaction (PCR) and its role in the determination of amplification efficiency, and then reviews the wide range of inhibitors and enhancers that can affect PCR efficiency. An optimised model system based on the amplification of three different sized DNA targets using both the LightCycler and a conventional thermal cycler was used to investigate the effects on amplification of two well known PCR inhibitors: haemoglobin, a protein constituent of blood that is often carried over in blood DNA purification processes; and phenol, an aromatic hydrocarbon routinely used in many DNA isolation techniques. In addition, the ability of known enhancers to overcome haemoglobin and phenol mediated PCR inhibition was investigated.
The work described suggests that the inhibition mechanisms of both haemoglobin and phenol involved the DNA polymerase and were not Taq DNA polymerase specific. From the results presented in this report the most likely mechanisms for haemoglobin inhibition and for phenol inhibition are the “irreversible ion-binding competition” theory and the “protein denaturation” theory respectively.
This report also illustrates that definitive patterns of inhibition and enhancement are not observed and that excessive enhancer addition can cause PCR inhibition. In addition, the use of a model system for investigating assay performance has proven to have an important role in the validation of the polymerase chain reaction.
It is hoped that an understanding of the mechanisms of action of two PCR inhibitors will enable workers in this field to account for unexpected results, to use suitable enhancers to improve the amplification and to scrutinise DNA extraction methods for suitability.