A Study On Polymorphism And Differentially Methylated Parental Allele Of Rs220030 In Snrpn Gene

Objective:To genotype SNP locus rs220030 which located in the promoter region of imprinted gene SNRPN by TaqMan SNP Genotyping Assay. Methylation-sensitive single-strand conformation polymorphism (MS-SSCA) method was used to detect the parental origin of allele in imprinted gene SNRPN. The potential feasibility of MS-SSCA was assessed for personal identification and paternity testing. Methods: We chose a robust tool, TaqMan SNP Genotyping Assay, to type rs220030 in 300 Chinese volunteers from Shanghai. After typing, the allele frequency and statistical parameters were assessed for forensic identification. Rs220030 (C/T) in maternal imprinted gene SNRPN was also typed by classical DGGE method in family trios. A new combination of DNA extraction and bisulfite conversion-QIAamp DNA Micro kit and EpiTect bisulfite kit-was used to extract and modify DNA from 3-mm dried blood spots. Then we used the MS-SSCA method to detect the parental origin of allele in imprinted gene SNRPN. Results:DNA of 300 Han individuals from Shanghai were genotyped for rs220030 by Taqman SNP Genotyping Assay. The genotype results and allele frequency were with 85 homozygous C/C,54 homozygous T/T and 161 heterozygous C/T. DGGE can also perform SNP typing for rs220030. The SNP rs220030 tested in this study was in Hardy-Weinberg equilibrium. This particular combination of DNA extraction and bisulfite conversion-using QIAamp DNA Micro kit and EpiTect bisulfite kit-results in successful bisulfite conversion when working with DNA isolated from dried blood spot microvolume samples. Methylated status of maternal and paternal allele was determined by MS-SSCA on four family trios with heterozygous children. Conclusion:Our data showed that rs220030 can combine with other SNPs which may reach the discrimination probability for forensic identification. DGGE method could also be utilized in single SNP locus typing due to its cheap and convenience. The new combination of efficient DNA extraction and bisulfite treatment provides high quality conversion of unmethylated cytosine to uracil for bisulfite genomic sequencing analysis of forensic stain samples. MS-SSCA had the potential application in the discrimination of differentially methylated parental allele of imprinted genes in forensic paternity testing.