印记基因5个SNP分型及亲代来源的检测

目的 采用复合PCR-Snapshot联合甲基化敏感酶切技术,检测印记基因中5个SNP的甲基化状态、印记亲代来源及分型.方法 选择15例亲子鉴定已证实为亲生关系的家系样本,采用单碱基延伸复合检测技术,检测家系样本IGF2AS rs1003483、SNURF rs220028、SNURF rs4906939、DLGAP2 rs6558478、SIM2 rs737380等5个SNP分型,同时选用核酸内切酶(McrBC)和甲基化敏感的限制酶(msRE) HhaⅠ、HpaⅡ消化子代DNA,验证印记基因的亲代来源.结果 经用本文方法检测,证实rs1003483为父源印记;rs220028、rs4906939为母源印记;rs6558478及rs737380未在差异甲基化区,不能确定其印记亲代来源.结论 复合PCR-Snapshot联合甲基化敏感酶切技术简单、高效,在检测多个SNP分型的同时可确定亲代来源,可在相关研究和实践中选用.     

采用Pyrosequencing和Pooling技术对SNP位点多态性分析

目的 建立基于pyrosequencing和Pooling技术进行SNP位点的法医学多态性分析技术.方法 对50名无关个体样本建立一适合pyrosequencing检测的组池;采用PyroMark Assay Design 2.0软件进行SNP位点等位基因定量分析的引物设计;对组池样本PCR产物进行焦磷酸测序检测.结果 检测的3个SNP位点多态性良好,其中位点rs220028与以往人群调查后频率数据无显著差异.结论 采用pyrosequencing和Pooling技术对SNP位点进行多态性分析,适合于位点的初筛及大规模群体调查.该技术准确可靠,方便快捷.     

采用Pyrosequencing和Pooling技术对SNP位点多态性分析

目的建立基于pyrosequencing和Pooling技术进行SNP位点的法医学多态性分析技术。方法对50名无关个体样本建立一适合pyrosequencing检测的组池;采用PyroMark Assay Design 2.0软件进行SNP位点等位基因定量分析的引物设计;对组池样本PCR产物进行焦磷酸测序检测。结果检测的3个SNP位点多态性良好,其中位点rs220028与以往人群调查后频率数据无显著差异。结论采用pyrosequencing和Pooling技术对SNP位点进行多态性分析,适合于位点的初筛及大规模群体调查。该技术准确可靠,方便快捷。     

中国朝鲜族H19基因上游差异甲基化区SNP分布

目的调查中国朝鲜族群体中H19基因上游差异甲基化区(differentially methylated region,DMR)SNP及单倍型分布,为法医学应用及群体遗传学研究提供基础数据。方法收集中国朝鲜族101份无关个体血样和14份来自5个亲缘关系已知的两代家系血样,用PCR-循环测序、McrBC消化DNA后PCR方法检测H19基因上游DMR的SNP,并用亲源印记等位基因(parentally imprinted allele,PIA)分型法进行单倍型检测,再计算相关遗传学参数。结果在H19基因上游DMR 1174bp的目的基因扩增产物中,共检出13个SNP(rs10840167、rs2525883、rs12417375、rs4930101、rs2525882、rs2735970、rs2735971、rs11042170、rs2735972、rs10732516、rs2071094、rs2107425、rs4930098)和5种单倍型,有9个SNP属于高鉴别能力的遗传标记,其单倍型具有较高的个人识别率,单倍型平均基因多样性(GD)为0.714。应用McrBC酶消化基因组DNA的PIA分型法确定了家系子代样本的母源单倍型。结论 H19基因上游DMR在中国朝鲜族群体中具有很高的遗传多态性,母源单倍型的确定进一步提高了印记基因的法医学鉴定效能。     

H19基因上游差异性甲基化区SNPs多态性研究

目的获得H19基因上游差异性甲基化区中SNPs的群体遗传学信息。方法采用PCR和测序技术,对105例中国北方汉族健康无关个体H19上游启动子区检测;使用Haploview 4.1和PowerStats V12软件进行统计学分析。选用甲基化敏感的限制内切酶(msRE)HpaⅡ,检测5个家系样本H19等位基因的亲代来源。结果测序结果显示,H19启动子区含有13个SNPs,组成5种单倍型,13种单倍型组合,其个体识别能力为0.856、多态性信息含量为0.67、非父排除率为0.498。经msRE HpaⅡ消化母源等位基因后,进行PCR及测序分析,检测出父源等位基因,排除1例和肯定4例家系的亲缘关系。结论 DNA甲基化标记和SNPs多态性检测,可同时进行多态性分型并确定等位基因的亲代来源,具有较高的法医学应用价值。     

DNA甲基化年龄推断模型在华东汉族人群中的跨平台应用

目的 评估基于DNA甲基化年龄推断模型在华东汉族人群中的法医学应用价值,为探索适用于不同检测平台的年龄推断模型提供理论依据。方法 根据已发表的中国汉族人群血液DNA甲基化年龄推断模型中6个年龄相关甲基化位点,使用焦磷酸测序和下一代测序（next-generationsequencing,NGS）技术分别检测48例样本的DNA甲基化水平,分别将其代入年龄推断模型后计算预测年龄,并与真实年龄进行比较。结果 两种检测技术下6个甲基化位点都与年龄相关,使用焦磷酸技术的R2为0.85,平均绝对误差（medianabsolutedeviation,MAD）为4.81岁,使用NGS技术的R2为0.84,MAD=4.41岁。结论 该血液DNA甲基化年龄推断模型可以在焦磷酸测序与基于NGS的多重目的区域甲基化富集测序技术下使用,并能够较为准确地推断年龄。     

片段长度差异等位基因特异性PCR——一种改良的SNP分型新方法

目的建立一种基于等位基因特异性PCR原理的改良SNP分型新方法:片段长度差异等位基因特异性PCR,并考察特异性引物的3'端第3位、第4位碱基错配对特异性延伸的影响。方法以SNP位点rs759117和rs760887为例,设计两条长度不同、3'末端分别与SNP两个等位基因碱基配对的上游引物,同时在两个等位基因特异性引物3'端第3或第4位碱基引入错配以增加特异性,下游为公用引物。PCR产物经聚丙烯酰胺凝胶电泳、银染显带后确定样本的基因型。结果不同SNP纯合子为长度不同的单一谱带,杂合子则为两条带,其结果与直接测序完全一致。两条特异性上游引物3'端第3或第4位碱基引入错配后非特异性延伸显著减少,且对PCR反应条件的严格性要求明显降低。结论片段长度差异等位基因特异性PCR是一种简单快速而有效的SNP分型新方法;两条特异性引物3'端第3、第4位碱基引入错配可使特异性显著增加     

基于DNA甲基化的三种常见体液组织来源鉴定

目的针对唾液、精液、外周血,找到一组可用于区分不同体液的组织差异性甲基化位点,为确定案件现场提取的体液组织来源提供科学依据。方法选取常见体液样本共49份,运用Illumina 850K甲基化芯片进行全基因组甲基化检测,筛选获得17个候选CpG位点;使用焦磷酸测序对55份样本进行测序,实际共检测了候选位点及其附近的位点共34个CpG位点。选择基于赤池信息量(AIC)的逐步条件多分类逻辑回归方法进行数据分析。结果基于3个CpG位点(cg25373595、cg18121066、cg17283169)构建了多分类逻辑回归模型用于体液组织来源预测,分别位于RAP1GAP2,TBCD,CALML3基因上。该模型对精液来源预测的AUC、灵敏度和特异性均为1,对唾液和静脉血来源的预测AUC和灵敏度均为1,特异性分别为0.99和0.98。在独立的唾液、精液和静脉血3种体液共24份样本中对该预测模型进行验证,预测结果全部正确。结论本文建立的基于3个CpG位点的体液组织来源鉴定方法可以实现唾液、精液和静脉血3种体液的有效区分,在法庭科学中具有潜在的应用价值。     

中国汉族人群不明原因猝死与NOS1AP基因多态性的相关性

目的研究日常活动中不明原因猝死(sudden unexpected death,SUD)者NOS1AP基因的单核苷酸多态性。方法收集60例一般日常活动中SUD病例心血样本作为SUD组,另外随机抽取80例无关个体的外周血样本作为对照组,提取基因组DNA,用特异性引物对NOS1AP部分SNP位点(rs10494366、rs10918859、rs12143842、rs12742393、rs3751284、rs348624)进行PCR扩增和直接测序,计算等位基因频率和基因型频率,并分析各SNP位点在SUD组与对照组之间的差异性。结果 NOS1AP第6外显子区域的rs3751284位点的等位基因频率和基因型频率在两组人群中的差异均有统计学意义(P0.05)。rs3751284位点的最小等位基因的频率在SUD组为0.325,在对照组为0.475。结论 rs3751284位点可能是SUD的易感基因位点。     

中国汉族人群不明原因猝死与NOSlAP基因多态性的相关性

目的研究日常活动中不明原因猝死（suddenunexpecteddeath,SUD）者NOSlAP基因的单核苷酸多态性。方法收集60例一般日常活动中SUD病例心血样本作为SUD组,另外随机抽取80例无关个体的外周血样本作为对照组,提取基因组DNA,用特异性引物对NOSlAP部分SNP位点（rsl0494366、rsl0918859、FSl2143842、rsl2742393、rs3751284、rs348624）进行PCR扩增和直接测序,计算等位基因频率和基因型频率．并分析各SNP位点在SUD组与对照组之间的差异性。结果NOSlAP第6外显子区域的rs3751284位点的等位基因频率和基因型频率在两组人群中的差异均有统计学意义（P〈0．05）。rs3751284位点的最小等位基因的频率在SUD组为0．325,在对照组为0．475。结论rs3751284位点可能是SUD的易感基因位点。     

DGGE技术检测小核核糖核蛋白多肽N基因rs220030位点的多态性

目的 分析小核核糖核蛋白多肽N(small nuclear ribonucleoprotein polypeptide N,SNRPN)基因SNP位点rs220030在中国汉族人群中的基因结构特征及多态性,获得群体遗传学资料.方法 应用变性梯度凝胶电泳(denatu^ng gradient gel electropho...     

Investigation of the methylation status around parent-of-origin detectable SNPs in imprinted genes

Methylation of CpG dinucleotides was investigated in five regions by bisulphite treatment of gDNA, PCR and cloning/sequencing. The gDNA was prepared from peripheral blood, saliva, semen, nails and hair from the head. In gDNA from peripheral blood, three regions were investigated in 16, 23 and 24 individuals, respectively (Fig. 2). In gDNA from other sources, three or five regions were investigated in five individuals (Fig. 3). In many of the sequenced fragments, all the CpG dinucleotides were either methylated or not, which support the idea that the parental origin of an allele may be determined by the methylation status of the allele. However, the methylation of CpG dinucleotides varies across the fragment in some of the sequenced fragments, especially from semen samples, which indicate that it may be difficult to determine the parental origin from some gDNA sources by restriction-enzyme analysis (DMPA method).     

利用RASSF1A位点检测母体血浆中胎儿SNP分型

目的探讨利用母体血浆中高甲基化RASSF1A位点进行胎儿SNP分型的应用价值。方法随机收集10个未孕健康妇女和45例不同孕期（早期5例、中期20例、晚期20例）孕妇的血样本及相应胎儿组织（绒毛组织、羊水、胎盘组织）;利用甲基化敏感限制性内切酶BstUI酶切后进行PCR,产物进行血细胞、血浆和胎儿组织（绒毛或胎盘）DNA RASSF1A序列的甲基化模式检测,并采用直接测序法对SNP rs4688725位点进行分型。结果经BstUI酶消化,RASSF1A序列在母体血细胞中均未检出,而在绒毛或胎盘组织中均能检出;在45名孕妇血浆中,RASSF1A序列均能被检出,且序列内的SNP分型与相应胎儿组织一致;在10名非孕妇女血浆中均未检出RASSF1A序列。结论母体和胎儿DNA中RASSF1A基因启动子区域的甲基化模式存在差异,可用于对母体血浆中的游离胎儿DNA进行SNP分型。     

华东汉族人群乙醇代谢酶相关SNP位点的多态性

目的对ADH2、ADH3、ALDH2和CYP2E1基因的40个SNP位点进行群体遗传学分析,得到多态性信息。方法利用PCR和质谱技术平台对SNP位点进行分型检测,通过对中国华东地区汉族人群199个无关个体的调查,统计分析40个SNP位点的等位基因分布频率。结果 40个SNP位点中,rs698、rs2241894（ADH3基因座）,rs13306164、rs671（ALDH2基因座）和rs28371746、rs2515641（CYP2E1基因座）的小等位基因分布频率（MAF）均大于1%,其它SNP位点的MAF均小于1%。结论 ADH2、ADH3、ALDH2和CYP2E1基因的40个SNP位点中,6个位点（rs698、rs2241894、rs13306164、rs671、rs28371746和rs2515641）在华东汉族人群中具有多态性。     

Application of fragment analysis based on methylation status mobility difference to identify vaginal secretions

Identifying vaginal secretions attaching or adhering to a suspect’s belongings would be beneficial for reconstructing the events that have taken place during a sexual assault. The present study describes a novel approach to identify vaginal secretions by fragment analysis using capillary electrophoresis, based on the mobility differences of PCR amplicons from bisulfite-treated DNA depending on methylation status. We targeted three genome regions including each of three vaginal secretion-specific methylated CpG sites reported previously: cg25416153, cg09765089, and cg14991487. In all three genome regions, the amplicon peaks for methylated genomic DNA (gDNA) sequences were only detected in vaginal samples, whereas samples of other body fluids (blood, saliva, semen, and deposit on skin surface) only showed amplicon peaks for unmethylated gDNA sequences. In vaginal secretions, the methylation ratio of each of the three targeted regions between samples was variable, while the ratios at the three regions in each sample were similar. Furthermore, commercial vaginal epithelial cells were completely methylated at the three regions. Therefore, vaginal secretion-specific methylation may derive from vaginal epithelial cells present in the sample.In forensic cases with a limited amount of DNA, the reproducibility of a detected peak using the present method is not high due to degradation of DNA by bisulfite treatment and subsequent stochastic PCR bias. However, it was possible to detect peaks from methylated DNA sequences by performing PCR and capillary electrophoresis in triplicate after bisulfite treatment, even when bisulfite treatment was performed using 0.5 ng of gDNA from vaginal secretions. In addition, the level of methylation at each targeted region was found to be stable in vaginal secretions stored for 1 year at room temperature. Therefore, we conclude that detection of the visual peak from vaginal secretion-specific methylated DNA sequence is useful to prove the presence of vaginal secretions. This approach has the potential to analyze multiple marker regions simultaneously, and may provide a new multiplex assay to identify various body fluids.     

Individual Identification of Racehorses from Urine Samples Using a 26‐Plex Single‐Nucleotide Polymorphism Assay

To construct a system for identifying individual horses from urine samples that are submitted for postracing doping tests, we developed a genotyping assay based on 26‐plex single‐nucleotide polymorphisms (SNPs). DNA was isolated from urine using a commercially available DNA/RNA extraction kit, and SNP genotyping was achieved with a SNaPshot^? technique. DNA profiles including 26 SNPs were acquired from urine samples and blood/hair samples. Within the studied Thoroughbred population, the 26‐plex assay showed a probability of identity of 5.80 × 10^?11. Compared to the conventional short tandem repeat assay, the SNP assay used less DNA, and the rate of successful genotyping was improved to 97% using aliquots of horse urine as small as 140 μL. The urinary DNA could be successfully genotyped under proper storage concerning refrigeration or freeze–thawing. This SNP assay can be used for individual identification when suspicious results are obtained from horse doping tests.     

中国汉族人群中Dia抗原和Dib抗原的分子遗传分析

目的研究中国汉族人群Diego血型系统中Dia和Dib抗原表达的分子遗传背景。方法采用血型血清学方法对2990例非血缘关系的捐血者进行Diego血型鉴定,从中随机选择20例表现型为Di(a-b )的样本,以及筛选到的所有Di(a b-)稀有血型样本,采用PCR-SSP、DNA直接测序方法分析Diego血型基因的分子遗传背景。结果2990例捐血者中,发现Di(a b-)表现型2例,Di(a b )167例,Di(a-b )2821例。随机选择的20例表现型为Di(a-b )的DNA样本,经PCR-SSP法检测的基因型为DI2DI2,对DI基因第19外显子直接测序,2561位上碱基为C。2例稀有血型Di(a b-)的DNA序列在19外显子2561位上碱基为T,基因型为DI1DI1。结论中国汉族人群Dia和Dib抗原表达的分子遗传基础是DI基因第19外显子2561位上碱基T-C的置换,引起第854位氨基酸的改变。