Enrichment Of Minority Alleles In Mixture By COLD-PCR

Background Mixture is one of the main challenging sample types usually meeting in forensic DNA lab. With the using of high-sensitive analysis methods and high-polymorphic, sex-linked genetic markers, our ability to acquire information form mixtures has increased greatly. However, even for those seasoned experts, some mix samples encountered in cases still represent great challenges. Presently, how to genotype the minority alleles in mixture still is one of the focuses in forensic DNA field.Objective To study the feasibility of Co-ampLification at Lower Denaturation Temperature PCR (COLD-PCR) for enrichment of minority alleles in mixtures, the COLD-PCR protocol for human Y-M175 Indel locus were developed, the typing results of regular PCR and COLD-PCR were compared and analyzed.Methods For 103 unrelated Han Chinese male, human Y-M175 locus was genotyped by a conventional PCR protocol developed in our lab. The mixtures were prepared by mixing the insertion and deletion DNA samples at certain ratio. To determine the critical denaturation temperature (Tc), the degree of heteroduplex subtraction at different temperature or melting temperature of amplicons were tested. Based on the Tc vlue determined, two COLD-PCR protocols were developed by insertion of two additional steps (PCR products hybridization and heteroduplex denaturation) into the conventional PCR cycles, one for the long amplicon based on PAGE-silver staining method, the other for the short amplicon based on real-time thermalcycler. The detectability of COLD-PCR and conventional PCR for minority alleles in mixtures were compared and analyzed.Results The frequency of Y-M175 insertion allele was 0.1650, while that of deletion0.8350. For Y-M175 long amplicon, the Tc was 76.4℃. For Y-M175 short amplicon, the Tm of insertion, deletion and heteroduplex were 79.9℃, 79.7℃and 78.2℃respectively. In COLD-PCR, the Tc of long and short amplicon were set at 76.4℃and 78.4℃respectively. For long amplicon, by COLD-PCR, the mix ratio at which reliable genotyping of minority allele can be achieved was 20:1, whereas it was not more than 5:1 by conventional PCR under the identical conditions. For short amplicon with real-time thermalcycler, the above mentioned ratio was 16:1 versus 64:1.Conclusions Without additional equipments and reagents required, COLD-PCR enabled the enrichment of minority alleles in mixtures, expanding the scope of typeable mixtures. The potential using of COLD-PCR in forensic mixture analysis deserves further study.