Assessment of high resolution melting analysis as a potential SNP genotyping technique in forensic casework

Abstract

High resolution melting (HRM) analysis is a simple, cost effective, closed tube SNP genotyping technique with high throughput potential. The effectiveness of HRM for forensic SNP genotyping was assessed with five commercially available HRM kits evaluated on the ViiA™ 7 Real Time PCR instrument. Four kits performed satisfactorily against forensically relevant criteria. One was further assessed to determine the sensitivity, reproducibility, and accuracy of HRM SNP genotyping. The manufacturer's protocol using 0.5 ng input DNA and 45 PCR cycles produced accurate and reproducible results for 17 of the 19 SNPs examined. Problematic SNPs had GC rich flanking regions which introduced additional melting domains into the melting curve (rs1800407) or included homozygotes that were difficult to distinguish reliably (rs16891982; a G to C SNP). A proof of concept multiplexing experiment revealed that multiplexing a small number of SNPs may be possible after further investigation. HRM enables genotyping of a number of SNPs in a large number of samples without extensive optimization. However, it requires more genomic DNA as template in comparison to SNaPshot®. Furthermore, suitably modifying pre-existing forensic intelligence SNP panels for HRM analysis may pose difficulties due to the properties of some SNPs.