Paper
An Investigation of PCR Inhibition Using Plexor®-Based Quantitative PCR and Short Tandem Repeat Amplification†,‡,§
Robyn E. Thompson M.S.,
George Duncan Ph.D.,
Bruce R. McCord Ph.D.,
Robyn E. Thompson M.S.
Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199
Search for more papers by this authorGeorge Duncan Ph.D.
Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199
Search for more papers by this authorCorresponding Author
Bruce R. McCord Ph.D.
Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199
Additional information and reprint requests:
Bruce McCord, Ph.D.
Department of Chemistry and Biochemistry
Florida International University
11200 SW 8th Street, CP304
Miami, FL 33199
E-mail: [email protected]
Search for more papers by this authorRobyn E. Thompson M.S.,
George Duncan Ph.D.,
Bruce R. McCord Ph.D.,
Robyn E. Thompson M.S.
Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199
Search for more papers by this authorGeorge Duncan Ph.D.
Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199
Search for more papers by this authorCorresponding Author
Bruce R. McCord Ph.D.
Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199
Additional information and reprint requests:
Bruce McCord, Ph.D.
Department of Chemistry and Biochemistry
Florida International University
11200 SW 8th Street, CP304
Miami, FL 33199
E-mail: [email protected]
Search for more papers by this author†
Presented in part at the 63rd Annual Meeting of the American Academy of Forensic Sciences, February 21–26, 2011, in Chicago, IL.
‡
Supported through NIJ grant # 2010-DN-BX-K204.
§
The points of view in the document are those of the authors and do not necessarily represent the official view of the US Department of Justice.
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.
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