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Article
An Investigation of PCR Inhibition Using Plexor®-Based Quantitative PCR and Short Tandem Repeat Amplification
Journal of Forensic Sciences
  • Robyn E. Thompson, Florida International University
  • George Duncan, Florida International University
  • Bruce McCord, Florida International University
Document Type
Article
Publication Date
11-1-2014
Keywords
  • forensic science,
  • DNA typing,
  • real-time polymerase chain reaction,
  • Plexor,
  • melt curve,
  • inhibition,
  • short tandem repeat
Abstract

A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the Plexor® real-time PCR quantification kit to evaluate PCR inhibition. This is performed by adding increasing concentrations of various inhibitors and evaluating changes in melt curves and PCR amplification efficiencies. Inhibitors examined included calcium, humic acid, collagen, phenol, tannic acid, hematin, melanin, urea, bile salts, EDTA, and guanidinium thiocyanate. Results were plotted and modeled using mathematical simulations. In general, we found that PCR inhibitors that bind DNA affect melt curves and CT takeoff points while those that affect the Taq polymerase tend to affect the slope of the amplification curve. Mixed mode effects were also visible. Quantitative PCR results were then compared with subsequent STR amplification using the PowerPlex® 16 HS System. The overall results demonstrate that real-time PCR can be an effective method to evaluate PCR inhibition and predict its effects on subsequent STR amplifications.

DOI
10.1111/1556-4029.12556
Citation Information
Robyn E. Thompson, George Duncan and Bruce McCord. "An Investigation of PCR Inhibition Using Plexor®-Based Quantitative PCR and Short Tandem Repeat Amplification" Journal of Forensic Sciences Vol. 59 Iss. 6 (2014) p. 1517 - 1529 ISSN: 0022-1198
Available at: http://works.bepress.com/george-duncan/2/