Improved Quantitation of Human DNA Using Quantitative Template Amplification Technology

Abstract

Enhancement of Q-TAT assay to detect PCR inhibitors and assess extent of DNA degradation in a forensic sample. Experiment completed through simultaneous detection of inhibition and degradation by adding a) SRY gene to Q-TAT to assess DNA degradation and gender identification and b) Renila luciferase pRL gene to detect PCR inhibitors. Q-TAT used to evaluate degraded DNA and detection of EDTA, hemin, humic acid and indigo inhibition in PCR. DNA estimations using modified Q-TAT compared to qPCR. Two internal standards were incorporated into the basic Q-TAT assay, SRY to assess degradation, and pRL to detect inhibition. An inverse correlation between amplicon size and amplification efficiency was observed in DNA degradation and PCR inhibition studies. Deviations from expected SRY: AMEL-Y ratio of 1.0, observed in modified Q-TAT assay for intact DNA, possibly reflecting degradation of DNA. The pRL amplicon proved to be a very sensitive detector of PCR inhibition.