Analysis Of Mitochondrial DNA Polymorphisms Based On Denaturing High-Performance Liquid Chromatography And Its Study In Forensic Application

Objective The purpose of this study is to establish a novelmethod for the detection of mitochondrial DNA (mtDNA) polymorphism based on denaturing high-performance liquid chromatography (DHPLC) and to explore the new mitochondrial DNA polymorphisms in coding region using DHPLC in order to improve the discrimination power of mtDNA in forensic DNA typing. Methods The primers were designed with primer3 software. mtDNA control region and coding region loci were amplified with the primers by the polymerase chain reaction (PCR). Using a technique of DNA pools and performing the analysis of pairwise combining samples, the DHPLC methods were evaluated and optimized. The methods established were employed to investigate the polymorphisms of mtDNA. Finally, a series of validation experiments were carried out to evaluate its utilities for forensic purpose. Results The DHPLC methods for analyzing two loci in mtDNA control region were established for the rapid identification of forensic case.Two methods based on DHPLC for exploring the polymorphisms in mtDNA coding region were also established. Using the two methods, we explored the polymorphism in the coding region sequence with covering 1435bp which was nominated as seven loci, were genotyped. The number of alleles observed at the seven loci ranged from 4 to 13, yielding 53 haplotypes in 120 unrelated individuals. The values of allele diversity for each locus ranged from 0.0782 (C locus) to 0.5701 (H locus). The haplotype diversity using all these loci was 0.8775. The validation experiments proved that the seven loci were suitable for forensic application. Conclusion The DHPLC methods were successfully established for forensic identification, and with high efficiency and low cost, they were expected to be important methods for forensic mtDNA identification. The new methods that we established for the investigation of polymorphisms in coding region loci were the foundation for exploring polymorphism in entire mitochondrial genome. Our study revealed that there were 53 haplotypes in the mtDNA coding region with covering 1435bp and seven loci and the haplotype diversity was 0.8775. Among these, four loci with higher diversity were proved to be suitable for forensic application and provided new genetic markers for the forensic mtDNA typing.