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

1.在H19基因上游5'区，共发现8个单倍型，包含14个snp，适用于差异甲基化亲本等位基因（DMPA）的检测方法。DMPA检测方法在8个印迹基因的新候选snp中应用失败。检测了H19、SNRPN、PEG3和MEST四个印迹基因的snp等位基因分布。因此，SNRPN和PEG3中H19FR和SNPs的单倍型多态性显示出更高的期望杂合性值。采用多重PCR和迷你测序法对上述3个位点加HYMAI SNP同时进行基因分型。4个位点的7个snp不仅在天然DNA中成功分型，而且在酶切DNA中也成功分型，用于多位点dmpa的鉴别。在克隆水平上，采用亚硫酸酯测序方法分析了5个印迹基因中多个snp周围CpGs的甲基化情况。一般情况下，根据基因的不同表达方式，父本或母本等位基因均可分为高甲基化或低甲基化。然而，测试区域的一些CpGs在几个克隆中表现出相反的甲基化状态。在PEG3克隆中，CpG甲基化的不一致性被观察到。

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.