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An Investigation of PCR Inhibition Using Plexor ® ‐Based Quantitative PCR and Short Tandem Repeat Amplification
Author(s) -
Thompson Robyn E.,
Duncan George,
McCord Bruce R.
Publication year - 2014
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/1556-4029.12556
Subject(s) - polymerase chain reaction , primer dimer , real time polymerase chain reaction , microbiology and biotechnology , chemistry , biology , multiplex polymerase chain reaction , gene , biochemistry
A common problem in forensic DNA typing is PCR inhibition resulting in allele dropout and peak imbalance. In this paper, we have utilized the P lexor ® 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 C T takeoff points while those that affect the T aq 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 P ower P lex ® 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.