Measuring human DNA degradation and gender detection in forensic DNA samples by q-PCR/HRM analysis

• Published in: Forensic science international. Genetics supplement series 2017-12, Vol.6, p.e552-e554
• Main Authors: Ginart, S., Caputo, M., Corach, D., Sala, A.
• Format: Article
• Language: English
• Subjects: Degraded DNA assessment; DNA quantitation; High resolution melting analysis; Male DNA detection
• ISSN: 1875-1768; 1875-175X
• DOI: 10.1016/j.fsigss.2017.09.211

Human DNA quantification, DNA degradation assessment and gender determination are key aspects in most field of human DNA analysis. The assay reported here is a tri-plex Real Time quantitative PCR reaction followed by high resolution melting (HRM) using Syto9 as intercalating dye. The system produces three amplicons: 1- transducin (beta)-like 1, Y-linked −TBL1Y (84bp), 2- DeGraded small target DNA–DGst- (152bp) and 3-DeGraded large target DNA-DGlt- (244bp). DNA quantitation is based on total fluorescence; TBL1Y amplicon allows detecting male DNA and the ratio DGst/DGlt to assess DNA degradation level. q-PCR quantitation proved good linearity in triplicates among 3.2pg/ul–50ng/ul DNA concentration range. Amplification efficiency (E) and reaction slope (m) mean values were 1.04 and 3.23 respectively. Upon HRM analysis, three melting peaks are detected in a male DNA sample and two if only female DNA is present. We define the parameter D as the ratio DGst/DGlt that reflects the extent of DNA degradation in a given sample. A direct correlation has been demonstrated between DNA damage and increased value of parameter D. This q-PCR approach is rapid, sensitive, and a cost-effective method suitable for detecting degraded DNA samples and applicable to any field where human DNA quantitation-qualification is required.