Title

An Investigation of PCR Inhibition Using Plexor®-Based Quantitative PCR and Short Tandem Repeat Amplification

Authors

Robyn E. Thompson, Florida International University
George Duncan, Florida International UniversityFollow
Bruce McCord, Florida International University

Document Type

Article

Publication Date

11-2014

Publication Title

Journal of Forensic Sciences

Keywords

forensic science, DNA typing, real-time polymerase chain reaction, Plexor, melt curve, inhibition, short tandem repeat

ISSN

0022-1198

Volume

59

Issue/No.

6

First Page

1517

Last Page

1529

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.

NSUWorks Citation

Thompson, Robyn E.; George Duncan; and Bruce McCord. 2014. "An Investigation of PCR Inhibition Using Plexor®-Based Quantitative PCR and Short Tandem Repeat Amplification." Journal of Forensic Sciences 59, (6): 1517-1529. doi:10.1111/1556-4029.12556.

DOI

10.1111/1556-4029.12556