A novel 16-plex DIP-SNP panel for forensic analysis of imbalanced DNA mixtures in a Han Chinese population

In the field of forensic genetics, the discovery of new genetic markers and the development of rapid detection techniques are of great significance for the identification of biological samples. The compound genetic marker DIP-SNP (deletion/insertion polymorphism-single nucleotide polymorphism) offers distinct advantages, including high polymorphism, the absence of stutter peaks, and a low mutation rate, making it a promising tool for the analysis of DNA mixtures. However, DIP-SNP genotyping based on capillary electrophoresis typically requires two separate amplification reactions, resulting in complex and time-consuming procedures. In this study, sixteen DIP-SNP markers were selected to develop a five-color fluorescent multiplex panel based on the amplification refractory mutation system (ARMS) and fragment length discrepant allele specific polymerase chain reaction (FLDAS-PCR) principles, enabling simultaneous genotyping in a single-tube reaction. In a Han Chinese population from Hubei province (n = 165), the effective number of alleles (A_e) ranged from 2.2160 to 2.9827 with a mean value of 2.6376, and the cumulative power of discrimination (CPD) and combined probability of exclusion (CPE) values were 0.999999999963 and 0.99830236, respectively, indicating that this panel is informative for personal identification and parentage testing in the studied population. In DNA mixture analysis, this panel successfully detected the minor component in two-person DNA mixtures at a ratio of 1:20 under optimized laboratory conditions with purified DNA. Our results suggest that this panel may serve as an effective supplement to conventional STR analysis in individual identification and parentage testing, and shows potential practical value in analyzing imbalanced DNA mixtures.