Investigation of PCR Inhibition Using Plexor (R)-Based Quantitative PCR and Short Tandem Repeat Amplification

• NCJ Number: 249387
• Author(s): Robyn E. Thompson, George Duncan, Bruce R. McCord
• Date Published: November 2014
• Length: 13 pages
• Agencies: NIJ-Sponsored
• Award: Detection and remediation of PCR inhibition using real time PCR melt curves as a diagnostic tool.

Annotation

This study used the Plexor® real-time PCR quantification kit to evaluate PCR inhibition, which is a common problem in forensic DNA typing, resulting in allele dropout and peak imbalance.

Abstract

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. The work was 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, the study 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. (Publisher abstract modified)