A multiplexed system for quantification of human DNA and human male DNA and detection of PCR inhibitors in biological samples

Purified genomic DNA from a male and a female individual were combined according to various ratios (see table) to mimic sexual assault evidence samples. The male gDNA was added to the mixtures at a constant concentration of approximately 25pg/μl. The mixtures were tested with the multiplex assay to determine the concentration of total and male DNA. The multiplex assay can quantify 25pg/μl of male DNA in the presence of up to approximately 25ng/μl of female DNA (1:1000 ratio). Electropherograms obtained by using the Yfiler^® PCR Amplification kit for the samples are shown: the male STR profile is complete up to the 1:1000 mixture ratio.

Human male genomic DNA extracted from whole blood was mixed with varying final concentrations of hematin: 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, and 40μM. 2.0μl of each DNA/hematin mix, containing 1.0ng of DNA, was quantified using the multiplex assay. The IPC Ct values were monitored (see bar graph). Partial inhibition was detected between 10 and 12.5μM hematin; total inhibition at 15μM hematin. 2.0μl of each DNA/hematin mix was also added to the Identifiler™ kit reactions. STR profiles from a subset of samples are shown. The results from the quantification assay provided reasonable predictions of samples that would fail STR analysis because of the presence of the PCR inhibitor (e.g., sample containing 15μM hematin). The ability of the assay to predict PCR inhibition was demonstrated by shifted IPC Ct values in the presence of increasing quantities of hematin. Similar results were obtained with samples containing increasing quantities of humic acid (data not shown).