Applicability of imprinted DIP-SNP markers in DNA mixture analysis: a pilot study

In recent years, a new compound genetic marker, deletion/insertion polymorphism-single nucleotide polymorphism (DIP-SNP), has been applied in forensic DNA mixture analysis. DIP-SNP genotyping based on capillary electrophoresis (CE) typically requires two separate amplification reactions and is difficult to interpret in complex DNA mixtures. In this study, four candidate imprinted DIP-SNP markers were selected to establish a multiplex polymerase chain reaction (PCR) set based on the amplification refractory mutation system (ARMS) principle for a one-tube reaction. Furthermore, parentally imprinted allele (PIA) typing was applied to selectively detect parental alleles. The imprinting patterns of rs35918685-rs185148 and rs11667883-rs76183558 were confirmed to be maternally imprinted, and both markers showed imprinting consistency in saliva, vaginal fluid, and menstrual blood. In DNA mixture analysis, these two markers successfully detected a minor DNA contributor in a two-person DNA mixture at a 1:20 ratio, indicating high analytical sensitivity for low template input and imbalanced mixtures. An improved RMNE method, described in a recent study, was applied to evaluate its effectiveness in narrowing down potential contributors in DNA mixtures, and a comparison of RMNE probabilities between conventional genotyping and PIA genotyping demonstrated the improved efficiency of PIA-based interpretation. These findings highlight the high forensic potential of imprinted DIP-SNP markers, particularly in the analysis of imbalanced DNA mixtures.