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

GINART SANTIAGO; CAPUTO, MARIELA; CORACH, DANIEL; ANDREA SALA

Seul

Congreso; 27th ISFG Congress, Seoul, South Korea; 2017

ISFG (International Society of Forensic Genetics)

Human DNA quantification, DNA degradation assessment and gender determination are key aspects in every field where human DNA analysis is required. This assay is a tetra-plex Real Time quantitative PCR reaction whose products are analyzed by HRM (high resolution melting) analysis using the intercalating dye SYTO9. The system produces four amplicons: 1- transducin (beta)-like 1, Y-linked -TBL1Y (84bp), 2- amelogenin (106/112 bp, female/male), 3- DeGraded small target DNA?DGst- (152bp) and 4-DeGraded large target DNA- DGlt- (244 bp). DNA quantitation is based on Amelogenin amplification, TBL1Y amplicon allows detecting male DNA and DGst/DGlt to assess DNA degradation level. Each fragment has different melting behavior reflected in different melting temperatures.The q-PCR system proved good linearity in triplicates among 16 pg/ul ? 50 ng/ul DNA concentration range. Adequate amplification efficiency and reaction slopes were obtained for all replicates. After HRM analysis, four melting peaks are detected in a male DNA sample and three melting peaks if only female DNA is present. The DGst / DGlt parameter reflects the integrity of the genetic material present in the analyzed samples. There is a direct correlation between DNA damage and increased ratio of DGst/DGlt. The ability to collect these data during quantification step enables to improve the marker selection. Additionally, the use of intercalating dyes (Syto9) considerably reduces the cost of analysis compared to other detection approaches.This q-PCR is rapid, sensitive, and a cost-effective method suitable for degraded DNA samples and applicable to any field where human DNA quantification is required.