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Analysis of the Effect of a Variety of PCR Inhibitors on the Amplification of DNA Using Real Time PCR, Melt Curves and STR Analysis

NCJ Number
249148
Author(s)
Bruce McCord; Arianna Pionzio; Robyn Thompson
Date Published
November 2014
Length
67 pages
Annotation
This project examined the effect of a variety of PCR inhibitors, using real time PCR, melt curve analysis, and STR typing in an effort to define the inhibitory mechanism of these materials and assist forensic analysts in interpreting their results.
Abstract
Examination of the effect of these inhibitors on internal control sequences used in real time PCR should assist designers of real time PCR quantification kits in developing better probes for inhibition. Overall, the results clarify the role of PCR inhibitors in PCR amplification and their effects on STR amplification. Three basic types of inhibitors were identified: DNA binding, polymerase binding, and mixed mode (inhibitors that affect both polymerase and template). Real time PCR amplification efficiency and melt curves assisted in identifying the modes of inhibition and also have some predictive power in defining downstream allele dropout. Although allele sequence had an effect on sensitivity to PCR inhibition, the length of the amplicon is most important. Individuals interested in validating new forensic methods are advised to carefully choose a range of inhibitors that encompass the range of modes of inhibition. The study examined the effects of simple treatments such as dilution, increasing polymerase, BSA, and magnesium concentration on the inhibitory effects. Also measured were the effects of different extractions on the success of removing inhibition. 6 figures, 5 tables, 16 references, and a listing of publications and events for the dissemination of the findings