NCJ Number
215143
Journal
Journal of Forensic Sciences Volume: 51 Issue: 4 Dated: July 2006 Pages: 795-804
Date Published
July 2006
Length
10 pages
Annotation
This study empirically demonstrates that, along with template DNA quantitation, the calculation of real-time polymerase chain reaction (PCR) amplification efficiencies can be used to identify samples that are contaminated by PCR inhibitors, which can lead to the loss of alleles from the larger short tandem repeat (STR) loci, and even cause complete false-negative results.
Abstract
This study shows that real-time PCR efficiency calculations can evaluate template DNA quality and identify problematic samples prior to genotyping or DNA sequence analysis. The Liu and Saint and the LinRegPCR methods provide comparable efficiency estimates and similar outlier detection results; however, optimal performance depends on adjusting the calculations to minimize the inclusion of baseline and linear-phase fluorescence value. Internal Positive Controls (IPC) can also be used to detect the presence of PCR inhibitors. The IPC is particularly useful in detecting false-negative results; however, it cannot be used to obtain a precise measure of inhibition severity when template samples are marginally compromised. Using the recommended rigorous procedures to identify tainted DNA samples offers an opportunity to consider inhibitor mitigation strategies prior to genotype or sequence analysis. Assays with varying concentrations of tannic acid were used to evaluate and adjust sample-specific amplification-efficiency calculation methods in order to optimize their inhibitor-detection capabilities. Kinetic outlier detection and prediction boundaries were calculated to identify amplification efficiency outliers. Sample-specific amplification efficiencies were calculated over a four-cycle interval starting at the threshold cycle to reliably detect the presence of 0.4 ng of tannic acid in a 25 ml PCR reaction. 2 tables, 7 figures, and 57 references