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

GINART, S.; CAPUTO, M.; CORACH, D.; SALA, A.

FORENSIC SCIENCE INTERNATIONAL.

ELSEVIER IRELAND LTD

Año: 2019 vol. 295 p. 1 - 7

0379-0738

We developed a q-PCR technique that simultaneously evaluates the extent of degradation anddetermines the gender of a human DNA donor. QYDEG HRM is a triplex real-time PCR whose products areanalysed by high-resolution melting (HRM). The system produces three amplicons: (1) transducin (beta)-like 1, Y-linked (TBL1Y) (84 bp); (2) large-target sequence (DGlt) (244 bp); and (3) small-target sequence(DGst) (152 bp). After HRM analysis, three melting peaks are detected in male DNA samples and two infemale DNA samples. An imbalance between the DGst and DGlt melting peak heights allows for theestimation of the extent of DNA degradation. For sensitivity assessment, triplicate aliquots of 0.0032 to50 ng/mL DNA were tested, denoting good linearity and reproducibility. The results also showed theanalysis to be precise and accurate in the DNA range of 0.04?5 ng/mL. Diverse types of DNA samples weretested: experimentally heat-degraded DNA; crime scene samples derived from casework and highlydegraded samples with partial STR profiles from corpse material and mass disaster events. The resultswere compared with those obtained from the Plexor1 and PowerQuant1 commercial kits. Additionally,the quantification results of the QYDEG HRM triplex correlate well with the STR amplification that wassubsequently obtained. The method is simple, cost-effective and helpful for determining the DNAintegrity and the sex of a sample donor in anyfield where human DNA quantification is required.