Assessment of reliability of parental allele detection method and its progressive application to the forensic fields

亲本等位基因检测方法的可靠性评估及其在法医学领域的逐步应用

• 批准号: 17590583
• 负责人: NAKAYASHIKI Nori
• 金额: $ 1.6万
• 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: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590583/
• 关键词: Forensic Medicine; Genomic Imprinting; Parent-of-Origin; DMR; DNA polymorphism; ゲノム・インプリンティング; 親判別; メチル化; クローニング

1.In-the region 5'upstream-of the H19 gene, eight haplotypes composed of 14 SNPs were discovered to be applicable to the detection method of differentially methylated parental allele (DMPA). Application of DMPA detection method to new candidate SNPs in eight imprinted genes was failed.2. Allele distributions of SNPs in four imprinted genes (H19, SNRPN, PEG3 and MEST) were examined. As a result, haplotype polymorphism of H19FR and SNPs in SNRPN and PEG3 revealed higher values of expected hetelozygosity. Multiplex PCR and mini sequencing method was used to simultaneous genotyping of above three loci plus HYMAI SNP. All seven SNPs in the four loci were successfully typed not only in native DNA but also in enzyme-digested DNA for discrimination of multilocus DMPA.3. Methylated situation of CpGs around several SNPs in five imprinted genes was analyzed by using bisulfite-sequencing method at clonal level. In general, either paternal or maternal allele was expectedly classified into highly methylated or hypo-methylated situation according to each expression manner of the gene. However, some CpGs in the tested region showed opposite methylation status in several clones. Inconsistency of CpG methylation was considerably observed in PEG3 clones.

1.在H19基因的5'上游区域，发现由14个SNP组成的8个单倍型适用于差异甲基化亲本等位基因（DMPA）的检测方法。 DMPA检测方法应用于8个印记基因的新候选SNP失败。 2．检查了四个印记基因（H19、SNRPN、PEG3 和 MEST）中 SNP 的等位基因分布。因此，H19FR 的单倍型多态性以及 SNRPN 和 PEG3 中的 SNP 揭示了更高的预期杂合性值。采用多重PCR和小型测序方法对上述三个位点加HYMAI SNP同时进行基因分型。四个基因座中的所有七个 SNP 不仅在天然 DNA 中成功分型，还在酶消化 DNA 中成功分型，用于区分多位点 DMPA.3。采用亚硫酸氢盐测序方法在克隆水平上分析了5个印记基因中多个SNP周围CpG的甲基化情况。一般来说，根据基因的每种表达方式，父系或母系等位基因预期被分为高甲基化或低甲基化情况。然而，测试区域中的一些 CpG 在几个克隆中显示出相反的甲基化状态。在 PEG3 克隆中明显观察到 CpG 甲基化的不一致。

项目成果

Investigation of discrimination method for DMPA(Differentially methylated parental allele)

DMPA（差异甲基化亲本等位基因）判别方法的探讨

• 发表时间: 2005
• 期刊: DNA polymorphism 13
• 作者: Nakayashiki N;Takamiya M;Aoki Y;Hashiyada M
• 通讯作者: Hashiyada M

DMPA (differentially methylated parental allele) 検出法に関する検討II

DMPA（差异甲基化亲本等位基因）检测方法研究二

• 发表时间: 2006
• 期刊: DNA多型 14
• 作者: 中屋敷徳;高宮正隆;黒瀬奈緒子;青木康博;橋谷田真樹
• 通讯作者: 橋谷田真樹

DMPA (differentially methylated parental allele)検出法に関する検討

DMPA（差异甲基化亲本等位基因）检测方法研究

• 发表时间: 2005
• 期刊: DNA多型 13
• 作者: 中屋敷徳;高宮正隆;青木康博;橋谷田真樹
• 通讯作者: 橋谷田真樹

Investigation of discrimination method for DMPA(Differentially methylated parental allele) III

DMPA（差异甲基化亲本等位基因）判别方法的探讨Ⅲ

• 期刊: DNA polymorphism 15 (in printing)
• 作者: Nakayashiki N;Takamiya M;Kumagai A;Aoki Y;Hashiyada M
• 通讯作者: Hashiyada M

DMPA (differentially methylated parental allele)検出法に関する検討III

DMPA（差异甲基化亲本等位基因）检测方法研究三

• 发表时间: 2007
• 期刊: DNA多型 15(印刷中)
• 作者: 中屋敷徳;高宮正隆;熊谷章子;青木康博;橋谷田真樹
• 通讯作者: 橋谷田真樹