Molecular assay for screening and quantifying dna in biological evidence: The modified Q-tat assay

A method is described for the quantitation of total human and male DNA. Q-TAT utilizes end-point, multiplex polymerase chain reaction (PCR) amplification of the amelogenin and SRY loci to quantify DNA and incorporates a cloned nonhuman template to detect PCR inhibition. Standard curves of fluorescence from amelogenin or SRY amplicons were generated from amplification of known amounts of NIST traceable SRM-female or SRM-male DNA. Curves showed good linearity up to 500 pg of SRM-template (R² > 0.99) and reliably estimated total and male DNA content in casework samples. The nonhuman pRL_null template included in each PCR was a sensitive indicator of known PCR inhibitors including EDTA, hemin, blue denim dye, and humic acid. Finally, the SRY amplicon was a sensitive indicator of male DNA and, in mixtures, could reliably estimate male DNA present in an excess of female DNA. The Q-TAT multiplex is a reliable quantitation method for forensic DNA typing.