title: An investigation of PCR inhibition using Plexor(®) -based quantitative PCR and short tandem repeat amplification.
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.
authors.affiliation: Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street, Miami, FL, 33199.
authors.forename: Robyn E
authors.initials: RE
authors.lastname: Thompson
authors.forename: George
authors.initials: G
authors.lastname: Duncan
authors.forename: Bruce R
authors.initials: BR
authors.lastname: McCord
mesh_terms: D001647:Bile Acids and Salts; D002130:Calcium Phosphates; D003094:Collagen; D016172:DNA Fingerprinting; D004492:Edetic Acid; D006146:Guanidines; D006427:Hemin; D006801:Humans; D006851:Hydrochloric Acid; D018895:Microsatellite Repeats; D008957:Models, Genetic; D010636:Phenols; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013634:Tannins; D013861:Thiocyanates; D014508:Urea
chemical_list: D001647:Bile Acids and Salts; D002130:Calcium Phosphates; D006146:Guanidines; D010636:Phenols; D013634:Tannins; D013861:Thiocyanates; C054436:guanidine thiocyanate; D006427:Hemin; D014508:Urea; D003094:Collagen; D004492:Edetic Acid; C485829:calcium phosphate, dibasic, anhydrous; D006851:Hydrochloric Acid
keywords: DNA typing; Plexor®; forensic science; inhibition; melt curve; real-time polymerase chain reaction; short tandem repeat