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

1.在H19基因上游5‘端，发现了由14个SNP组成的8种单倍型，可用于亲本差异甲基化等位基因(DMPA)的检测。结论：1.DMPA检测方法在8个印迹基因新候选SNPs检测中的应用失败。检测了H19、Snrpn、Peg3和MEST四个印记基因的SNPs等位基因分布。因此，在Snrpn和Peg3中，H19FR和SNPs的单倍型多态显示出较高的预期杂合度。采用多重聚合酶链式反应和微测序方法对上述3个基因座和HYMAI SNP同时进行基因分型。这四个基因座上的7个SNP不仅在天然DNA中成功地分型，而且在酶切DNA中也成功地进行了分型，以区分多位点DMPA。用亚硫酸氢盐测序法在克隆水平上分析了5个印迹基因中多个SNPs周围的CpGS甲基化情况。一般来说，父亲或母亲的等位基因根据基因的表达方式被意外地分为高度甲基化或低甲基化状态。然而，受试区的一些CPGS在几个克隆中表现出相反的甲基化状态。在Peg3克隆中观察到CpG甲基化的不一致性。

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.