title: The effect of internal control sequence and length on the response to PCR inhibition in real-time PCR quantitation.
abstract: PCR inhibitors can originate from a variety of sources and can co-extract with the DNA template, resulting in reduced amplification and/or dropped alleles. Currently real time PCR is used to provide a check for the presence of PCR inhibition by monitoring the quality of amplification of an internal control. In this paper we examine the effect of internal control length and sequences on its sensitivity to PCR inhibition by varying concentrations of commonly encountered PCR inhibitors. Data from both amplification and melt curves were evaluated. The results show that while amplicon sequence has minor effects on amplification efficiency and melt curves, amplicon length has a more dramatic effect, regardless of inhibitor type. Given the increasing variety of STR typing kits and their documented differences in performance with respect to inhibition, the data obtained in this study can be used to assist designers of real time PCR kits to adjust their internal PCR controls (IPC) to permit a more targeted estimation of inhibition.
authors: Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street CP304, Miami, FL 33199, USA.
authors: Arianna M
authors: AM
authors: Pionzio
authors: Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th Street CP304, Miami, FL 33199, USA. Electronic address: mccordb@fiu.edu.
authors: Bruce R
authors: BR
authors: McCord
mesh_terms: D001647:Bile Acids and Salts; D003094:Collagen; D003596:Cytosine; D016172:DNA Fingerprinting; D019791:Guanidine; D006147:Guanine; D006801:Humans; D006812:Humic Substances; D008543:Melanins; D018895:Microsatellite Repeats; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA; D013634:Tannins; D014508:Urea
chemical_list: D001647:Bile Acids and Salts; D006812:Humic Substances; D008543:Melanins; D013634:Tannins; D006147:Guanine; D003596:Cytosine; D014508:Urea; D003094:Collagen; D019791:Guanidine
keywords: DNA typing; Forensic science; Inhibition; Plexor; Real-time PCR; Short tandem repeats