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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
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)
Date Published: November 1, 2014