Maura Barbisin, Rixun Fang, Manohar R Furtado and Jaiprakash G Shewale
Forensic analysts routinely encounter samples containing mixtures of DNA from male and female contributors and PCR inhibitors due to exposure to environmental insults. In order to select the appropriate STR analysis methodology for such samples and obtain optimal results at first pass, it is desirable to determine the relative quantities of male and female DNA and to detect the presence of PCR inhibitors at an early stage in the sample processing workflow. Here we describe a multiplex real-time PCR assay that can provide the desired information in a single reaction. Briefly, the simultaneous quantification of human and human male DNA is achieved by measuring the RPPH1 human target and the SRY male-specific target. At the same time a synthetic sequence is co-amplified as an Internal PCR Control (IPC) to detect the presence of PCR inhibitors. The assay has a good dynamic range (0.023–50 ng/μL) and can detect 25 pg/μL of human male DNA in the presence of ten thousand-fold excess of human female DNA. In addition, the ability of the assay to predict PCR inhibition was demonstrated by shifted IPC C T values in the presence of increasing quantities of hematin. All the real-time PCR results showed a good correlation with the downstream STR profiles obtained from a large set of various sample types therefore demonstrating that this assay can be considered a guiding tool to predict the performance of the STR genotyping kits with forensic samples.
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