Genotyping genetic markers from LCN and degraded DNA by HRM and their application in hair shaft

Degraded and low copy number (LCN) DNA samples are common challenging materials in forensic casework because they increase the difficulty of sample processing and reduce the possibility of obtaining genetic information from DNA. High-resolution melting (HRM) curve analysis is promising for genotyping genetic markers and has been applied to the detection of LCN and degraded DNA in the field of forensic science. However, the exact assessment based on HRM at multiple genetic markers from both degraded and LCN DNA materials has not been optimized. To explore the ability of HRM to genotype LCN and degraded DNA samples, we selected three genetic markers to genotype in experimental LCN and degraded DNA and practical hair shaft materials, which are often encountered as degraded and LCN DNA in forensic medicine. The results show that DNA samples of as low as 100 pg and as short as 60 bp were successfully genotyped by the HRM assay at all three genetic markers, whereas in hair shaft DNA, two loci were accurately genotyped. The HRM assay established in this study can be applied to LCN and degraded DNA analysis in forensic casework and can act as a reference point before genotyping short tandem repeat markers. Developing the HRM strategy for genotyping DNA genetic markers enriches detectable targets in hair shaft samples and provides valuable data for further exploration.