Development Of A Multiplex Methylation-sensitive Restriction Enzyme-based SNP Typing System For Deconvolution Of Semen-containing Mixture

Objective:We used semen-specific methylation sites to confirm the presence of semen in mixed stains,and at the same time used semen-specific methylation sites combined with SNP sites to confirm population polymorphism which can be used for individual identification of semen donors in mixed stains.Methods:In this study,we first used methylation sensitive restriction endonuclease(MSRE)to digest the genomic DNA,and then used multiplex PCR and SNaPshot technique to detect semen-specific methylation sites and their combined SNPs.A total of 12 semen-specific Cp G-SNP regions were constructed,including 12 methylation sites and 16 SNP sites,and we also detected one tissue-specific methylation site of saliva,blood,and vaginal secretion,respectively.The methods are as follows:(1)tissue specific methylation sites were selected from literatures,and SNPs were searched within 400 bp upstream and downstream of methylation sites using common SNP 151 dataset of UCSC database.(2)Primer 3 software was used to design primers for the selected Cp G-SNP regions.There was only one recognition site(Hha Ⅰ,Hpa Ⅱ,HpyCH4 Ⅳ and BstU Ⅰ)in every Cp G-SNP region.(3)The designed primers were used for tissue specificity verification.(4)All primers were used for multiplex amplification,and then we optimized the multiplexed amplification system.(5)15 methylation loci and 16 SNP loci within 12semen-specific Cp G-SNP regions were detected by SNaPshot technique,and we obtained the genotyping of semen donor.(6)The forensic parameters of the constructed system were calculated.Results:1.A multiplex detection system that contained 3 tissue specific methylation sites of saliva,blood,vaginal secretion and 12 semen-specific Cp G sites and 16 polymorphic SNPs are successfully constructed.2.The cumulative discrimination power of the system in the northern Chinese Han population is 0.9998.3.Samples could be successfully identified through our multiplex detection system when the purified DNA concentration was equal to or greater than 10 ng.4.Using this system,10 ng of total DNA and DNA mixture with semen content up to 50% could be typed successfully.5.The source of the tissue in the mixed stain composed of semen,blood,saliva,and vaginal secretions can be successfully distinguished and the SNP typing of the semen donor can be obtained.Conclusion:We have developed a method to detect semen,saliva,blood and vaginal secretion in mixed stains,and to obtain the semen donor typing at the same time.