组织特异性甲基化及其连锁SNP在混合斑鉴定中的应用研究

Detection and identification of mixed stains has always been a difficult problem in forensic cases. The methods now always used are detection and interpretation of mixed STR results, detection of different body fluid component and semen unique genetic markers and so on. The use scope of these strategies are greatly restricted due to the following factors，extremely complex explaination of the results, sex restriction, requirement of expensive instrument and professional operation and analysis persons. In recent years, the DNA methylation methods based on the differential methylation of each tissue or fluid are used for fluid identification gradually. This study intends to establish a compound detection system based on the linkage relationship of DNA methylation markers and SNPs. We will firstly distinguish different fluids through the following procedures, digesting genomic DNA with methylation sensitive restriction enzyme, amplifying tissue specific expression genes with fluorescence labeling primers, capillary electrophoresis and professional software analysis. Then we will detect the alleles of SNPs which chained with the methylation markers using the SNaPshot micro sequencing technology. We will finally obtain the individual information of the body fluid provider through multiple DNA methylation-SNP linked markers. This detection system distinguishes different body fluids through the characteristics of differential methylation of DNA in different tissues and carrying individual identification through SNPs linked with methylation markers. This test method is not restricted by fluid types and can fully utilize the present forensic STR analysis platform, This will bring great application prospect for forensic mixed DNA detection.

混合斑的检验及鉴定一直是法医案件侦破中的难题。目前常用于混合斑检验的策略有混合STR分型、不同体液的成分分离、精液特有的遗传标记检测等。这些策略由于结果解释极其复杂、受性别限制、需要昂贵的仪器及专业的操作和分析人员等因素使其使用范围大大受限。近年来，基于组织特异的DNA甲基化检测方法逐渐用于体液鉴别。本项目拟构建甲基化-SNP复合检测体系以鉴定混合斑。运用甲基化敏感酶消化DNA样品，用荧光标记的引物复合扩增组织特异表达的基因，经毛细管电泳区分不同组织的样品；运用SNaPshot微测序技术对与甲基化标记基因连锁的SNP分型，通过对多个甲基化-SNP连锁基因的检测最终得到体液提供者的SNP分型图谱。该检测体系利用甲基化的组织特异性特征先把不同来源的体液区分开，再利用连锁的SNP进行个人识别。这种检测方法不受体液来源限制且可完全利用目前法医STR分型平台，将给法医混合斑检验带来很大的应用前景。

混合斑的检验及鉴定一直是法医案件侦破中的难题，它主要包括体液来源鉴定及个体识别两个方面。目前对于案发现场常见单一体液的来源检验以及鉴别技术相对成熟，但多存在诸如特异性差、灵敏度低、对样品有损坏、检测的生物分子不稳定等缺陷，对混合体液几乎束手无策。近年来，基于组织特异的DNA甲基化检测方法逐渐用于体液鉴别。常用于混合斑个体识别的策略有混合STR分型、不同体液的成分分离、精液特有遗传标记检测等。这些策略由于结果解释极其复杂、受性别限制、需要昂贵的仪器设备及专业的操作和分析人员等因素使其使用范围大大受限。本项目拟构建基于组织特异性DNA甲基化的SNP分型体系以同时进行混合斑的体液来源鉴定与个体识别。运用甲基化敏感限制性内切酶结合PCR的方法鉴别体液样品的来源；运用SNaPshot微测序技术对与组织特异性甲基化标记（CpG）相邻的多态性SNP分型；通过对多个CpG-SNP复合标记的检测最终得到体液提供者的SNP分型图谱。本项目通过对常见体液的特异性甲基化标记筛选及其组织特异性验证、甲基化敏感限制性内切酶识别序列筛选、与组织特异性CpG相邻的SNP筛选及其多态性验证、CpG-SNP复合检测体系构建以及复合检测体系的法医学参数检验等步骤成功建立了一套基于精液特异性甲基化标记的SNP分型体系。通过12个精液特异的CpG以及三个血液、唾液和阴道分泌物特异的CpG标记，该体系可成功检测由精液、血液、唾液和阴道分泌物四种常见体液构成的混合斑。此外，通过与12个CpG相邻的16个多态性SNP，该体系可对精液供体进行个体识别，其在中国汉族人群中的累积个体识别概率大于0.9998。该体系对于模板DNA≥10 ng或者精液含量＞10%的混合斑均可成功分型。该研究为法医实践中同时进行混合斑的体液来源鉴定和个体识别提供了一个非常有价值的策略。