Detection Modification Polymorphism on DNA Methylation and its application to Forensic Investigation

DNA甲基化修饰多态性检测及其在法医侦查中的应用

• 批准号: 12670403
• 负责人: NAKAYASHIKI Nori
• 金额: $ 1.34万
• 依托单位: Iwate Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-12670403/
• 关键词: polymorphism; genomic imprinting; DNA methylation; Legal Medicine; 多型; メチレーション

Regions containing SNPs in H19 (maternal expression) and IGF2 gene (paternal expression), which are involved in an imprinting domain on 11p15.5, were investigated concerning existence of diversity on methylation status and selective detection of parental allele in DNA from normal peripheral blood.At first, we examined known SNPs in H19 and IGF2 gene in order to determine detection methods and find allele distributions. In addition, a haplotype polymorphism composed of three SNPs close to H19 SNP, named H19FR, was analyzed by PCR-SSCP, ARMS PCR and PCR-CDGE, and three major haplotypes with allele frequencies of 0.5177, 0.3409 and 0.1414 were observed.After treatment of genomic DNA with bisulfite reagent, a methylated allele of H19FR with heterozygous genotype was selectively amplified by methylation-specific PCR. However, polymorphic methylation status in CpGs was not observed in this study.As another strategy for recognizing parental origin, genomic DNA was treated with methylation-sensitive restriction enzyme and followed by H19FR or H19 SNP typing. As a result, DNA sample having heterozygous genotype in either system showed a single allele derived from methylated paternal origin. The IGF2 SNP revealed, however, only 27 % of maternal allele after the same procedure for H19 SNPs. It is assumable that this method applied for SNPs in upstream region of H19 will be a useful tool for forensic examination to discriminate parental origin.

本研究对11p15.5上的印记结构域H19（母本表达）和IGF 2（父本表达）基因中的SNPs区域进行了研究，旨在探讨正常人外周血DNA甲基化状态的多样性和亲本等位基因的选择性检测。此外，通过PCR-SSCP、ARMS PCR和PCR-CDGE分析了由三个与H19 SNP接近的SNP组成的单倍型多态性，命名为H19 FR，观察到三种主要单倍型，其等位基因频率分别为0.5177、0.3409和0.1414。基因组DNA经亚硫酸氢盐试剂处理后，甲基化特异性PCR选择性扩增H19 FR基因杂合型甲基化等位基因。作为识别亲本来源的另一种策略，基因组DNA经甲基化敏感的限制性内切酶处理，然后进行H19 FR或H19 SNP分型。结果，在任一系统中具有杂合基因型的DNA样品显示出源自甲基化父系来源的单个等位基因。然而，IGF 2 SNP在H19 SNP的相同程序后仅显示27%的母体等位基因。该方法应用于H19基因上游区域的SNPs分析，可作为法医学鉴定亲本来源的有效工具。