A comparison of the effects of PCR inhibition in quantitative PCR and forensic STR analysis

Abstract

In this paper we compare the effects of three representative PCR inhibitors using quantitative PCR (qPCR) and multiplex STR amplification in order to determine the effect of inhibitor concentration on allele dropout and to develop better ways to interpret forensic DNA data. We have used humic acid, collagen and calcium phosphate at different concentrations to evaluate the profiles of alleles inhibited in these amplifications. These data were correlated with previously obtained results from quantitative PCR including melt curve effects, efficiency changes and cycle threshold (Ct) values. Overall, the data show that there are two competing processes that result from PCR inhibition. The first process is a general loss of larger alleles. This appears to occur with all inhibitors. The second process is more sequence specific and occurs when the inhibitor binds DNA, altering the cycle threshold and the melt curve. This sequence-specific inhibition results in patterns of allele loss that occur in addition to the overall loss of larger alleles. The data demonstrate the applicability of utilizing real-time PCR results to predict the presence of certain types of PCR inhibition in STR analysis.

Characterization of degradation and heterozygote balance by simulation of the forensic DNA analysis process

“…8). Inhibition of the Taq polymerase reduces PCR efficiency and increases the imbalance, the effect is greatest on high molecular weight fragments [40, 41]. Therefore the effect of inhibition is the same as degradation.…”

A simple, rapid, cost-effective and sensitive method for detection of Salmonella in environmental and pecan samples

“…; Funes‐Huacca et al . ), thus competing with cofactors responsible for the activity of enzyme, or may bind with other reaction components such as dNTPs or DNA template, thereby inhibiting PCR amplification. There are several published DNA extraction/preparation and purification methods for removing inhibitors from environmental samples, but these methods are time‐consuming or expensive, limiting their use when handling a large number of samples (Stauffer et al .…”

“…Impairment of direct DNA amplification by PCR from an environmental sample occurs due to naturally occurring PCR inhibitors such as bile salts and complex polysaccharides in feces (Lantz et al 1997;Monteiro et al 1997), humic substances in soil (Kozdr oj and van Elsas 2000) and tannins on the surfaces of nuts (pecan) (Krause et al 2001) that could be coextracted with the DNA template (Akane et al 1994;Eilert and Foran 2009). PCR inhibitors may directly inactivate Taq polymerase by binding to its active site (Opel et al 2009;Funes-Huacca et al 2011), thus competing with cofactors responsible for the activity of enzyme, or may bind with other reaction components such as dNTPs or DNA template, thereby inhibiting PCR amplification. There are several published DNA extraction/preparation and purification methods for removing inhibitors from environmental samples, but these methods are time-consuming or expensive, limiting their use when handling a large number of samples (Stauffer et al 2008).…”

The Development of Direct Ultra-Fast PCR for Forensic Genotyping Using Short Channel Microfluidic Systems With Enhanced Sieving Matrices

“…While this type of rtPCR monitoring may be susceptible to primer dimers, these do not interfere with the amplification and typically take place during the plateau stage. 66 Both Taqman and intercalating dye real time systems have been shown to be accurate and produce similar results. 67 The last type of probe commonly used in forensics is the Plexor quantification system designed by Promega Corporation.…”