Human DNA degradation assessment and male DNA detection by quantitative-PCR followed by high-resolution melting analysis.

We developed a q-PCR technique that simultaneously evaluates the extent of degradation and determines the gender of a human DNA donor. QYDEG HRM is a triplex real-time PCR whose products are analysed by high-resolution melting (HRM). The system produces three amplicons: (1) transducin (beta)-like 1, Y-linked (TBL1Y) (84bp); (2) large-target sequence (DGlt) (244bp); and (3) small-target sequence (DGst) (152bp). After HRM analysis, three melting peaks are detected in male DNA samples and two in female DNA samples. An imbalance between the DGst and DGlt melting peak heights allows for the estimation of the extent of DNA degradation. For sensitivity assessment, triplicate aliquots of 0.0032 to 50ng/μL DNA were tested, denoting good linearity and reproducibility. The results also showed the analysis to be precise and accurate in the DNA range of 0.04-5ng/μL. Diverse types of DNA samples were tested: experimentally heat-degraded DNA; crime scene samples derived from casework and highly degraded samples with partial STR profiles from corpse material and mass disaster events. The results were compared with those obtained from the Plexor® and PowerQuant® commercial kits. Additionally, the quantification results of the QYDEG HRM triplex correlate well with the STR amplification that was subsequently obtained. The method is simple, cost-effective and helpful for determining the DNA integrity and the sex of a sample donor in any field where human DNA quantification is required.