Y-STR genetic screening by high-resolution melting analysis

Abstract

ABSTRACT. Currently, the widely used automated capillary electrophoresis-based short tandem repeat (STR) genotyping method for genetic screening in forensic practice is laborious, time-consuming, expensive, and technically challenging in some cases. Thus, new molecular-based strategies for conclusively identifying forensically relevant biological evidence are required. Here, we used high-resolution melting analysis (HRM) for Y-chromosome STR genotyping for forensic genetic screening. The reproducibility of the melting profile over dilution, sensitivity, discrimination power, and other factors was preliminarily studied in 10 Y-STR loci. The results showed that HRM-based approaches revealed more genotypes (compared to capillary electrophoresis), showed higher uniformity in replicate tests and diluted samples, and enabled successful detection of DNA at concentrations as low as 0.25 ng. For mixed samples, the melting curve profiles discriminated between mixed samples based on reference samples with high efficiency. The triplex Y-chromosome STR HRM assay was performed and provided a foundation for further studies such as a multiplex HRM assay. The HRM approach is a one-step application and the entire procedure can be completed within 2 h at a low cost. In conclusion, our findings demonstrate that the HRM-based Y-STR assay is a useful screening tool that can be used in forensic practice.

Application of PCR-HRM method for microsatellite polymorphism genotyping in the LDHA gene of pigeons (Columba livia)

“…HRM analysis is a one-tube and post-PCR method based on the intercalation of fluorescence dye into double-strand DNA. Fluorescence changes during dsDNA melting enable to discriminate genotypes [ 22 ]. High-resolution melting analysis is a relatively easy and rapid (the entire procedure can be completed in 1.5–2 hours), one-step, cost-effective application [ 22 , 41 , 42 ].…”

“…Fluorescence changes during dsDNA melting enable to discriminate genotypes [ 22 ]. High-resolution melting analysis is a relatively easy and rapid (the entire procedure can be completed in 1.5–2 hours), one-step, cost-effective application [ 22 , 41 , 42 ]. Moreover, it was found that STRs analysed by HRM had more genotypes than in capillary electrophoresis [ 22 , 41 , 42 ].…”

“…High-resolution melting analysis is a relatively easy and rapid (the entire procedure can be completed in 1.5–2 hours), one-step, cost-effective application [ 22 , 41 , 42 ]. Moreover, it was found that STRs analysed by HRM had more genotypes than in capillary electrophoresis [ 22 , 41 , 42 ]. Demonstrated in this study method gave possibility to rapid discrimination of the (TTTAT) 3-5 microsatellite polymorphism in the pigeon’s LDHA gene, that may serve as an rapid test for the commercial genotyping services.…”

“…Moreover, data obtained from microsatellite analyses have confirmed that individual microsatellites tend to be more polymorphic, and thus more informative, providing greater resolution in genetic studies than other molecular markers, including SNPs [ 21 ]. For example, Y-chromosome short tandem repeats (Y-STRs) are powerful human genome marker and have been used for many years in forensic practice [ 22 ]. The traditional approach used in microsatellites genotyping procedures is based on locus-specific polymerase chain reaction (PCR) for product amplification and polyacrylamide gel electrophoresis for genotypes identification [ 22 ].…”

“…For example, Y-chromosome short tandem repeats (Y-STRs) are powerful human genome marker and have been used for many years in forensic practice [ 22 ]. The traditional approach used in microsatellites genotyping procedures is based on locus-specific polymerase chain reaction (PCR) for product amplification and polyacrylamide gel electrophoresis for genotypes identification [ 22 ]. Since the late 1990s, short tandem repeats (STRs) have been analysed using capillary electrophoresis (CE)–an automated technique utilizing laser-induced fluorescence to identify PCR products (amplicons are generated using fluorescence-labelled primers).…”