Portions of this paper were presented at the 60th Annual Meeting of the American Academy of Forensic Sciences, February 18–23, in Washington, DC.
A Study of PCR Inhibition Mechanisms Using Real Time PCR
Article first published online: 10 DEC 2009
© 2009 American Academy of Forensic Sciences
Journal of Forensic Sciences
Volume 55, Issue 1, pages 25–33, January 2010
How to Cite
Opel, K. L., Chung, D. and McCord, B. R. (2010), A Study of PCR Inhibition Mechanisms Using Real Time PCR. Journal of Forensic Sciences, 55: 25–33. doi: 10.1111/j.1556-4029.2009.01245.x
This project was supported under award 2006-DN-BX-K006 from the National Institute of Justice, and by the Florida International University Dissertation Year Fellowship (2008). Points of view in the document are those of the authors and do not necessarily represent the official view of the U.S. Department of Justice.
- Issue published online: 4 JAN 2010
- Article first published online: 10 DEC 2009
- Received 3 Sept. 2008; and in revised form 24 Dec. 2008; accepted 27 Jan. 2009.
- forensic science;
- DNA typing;
- real time polymerase chain reaction;
- humic acid;
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.