常染色体21个SNPs多态性分型方法研究

目的建立常染色体21个SNPs的多态性分型方法。方法采用荧光标记公用引物和等位基因特异性引物原理设计SNP复合扩增引物体系,对45个备选SNP位点筛选,选出21个及性别Amelogenin构成复合扩增体系。PCR产物经3130XL型电泳仪电泳分离,GeneMaperTM3.0数据分析软件分析结果。同时随机选取6份样品,使用测序方法对SNP分型并进行测序验证。结果应用本研究建立的复合扩增体系扩增样品,产物经毛细管电泳后,每个SNPs均可正确判定基因型。随机选取6份样品SNPs位点测序结果显示,荧光标记SNPs复合扩增分型与直接测序结果完全一致。结论本研究建立的荧光标记公有引物特异性片段常染色体21个SNPs复合扩增方法是SNP多态性分析的一种有效方法,并有助于解决SNP分型识别能力、效率、通量和高成本的问题。     

高分辨率熔解曲线分析技术及其在法医学中的应用

高分辨率熔解曲线(high-resolution melting,HRM)分析技术是一种利用DNA杂交与退火熔解特性进行核酸分析的技术,主要通过对DNA解离活动中荧光染料变化的监控,实现突变的筛查和检测。HRM分析技术不需要探针标记,并且能够在很大程度上减少测序的负担,具有特异性好、通量高、时间成本低、便于操作等优点。HRM分析技术还是一种同质闭管的检测方法,聚合酶链式反应(polymerase chain reaction,PCR)与后续检测均在同一孔内完成,无需转移样品,能有效的减少交叉污染的可能性。近年来有越来越多的专家学者尝试将HRM分析技术应用于法医学研究和相关案例中。本文拟对高分辨率熔解曲线分析技术的原理、技术特点、局限性以及在法医学中的应用进行综述。     

Y—STR基因座分型缺失分析

目的分析Y—STR基因座等位基因分型缺失数据,为法医学提供应用参考。方法收集浙江汉族4477名无关男性个体血样,自动工作站磁珠法提取DNA,Y—filer^TM试剂盒进行复合扩增,Gene Mapper IDv3．2分析软件分析Y-STR数据,统计出现基因分型缺失的概率。结果在4477名无关个体的Y—STR数据中,有来自23种单倍型的26个样本Y-STR分型各有1个短片段基因座的基因分型缺失,而其它长片段基因座的分型均完全正常。基因分型缺失的发生频率为0．518％。结论Y-STR基因座分型缺失具有一定的发生率,在日常检案中应注意防止误判。     

Y染色体微缺失和突变的全同胞关系鉴定

目的探讨Y染色体微缺失和突变时,两男性个体间的全同胞关系鉴定。方法提取两样本DNA,检测Y-STR分型及常染色体STR分型,通过IBS法、ITO法及全同胞-无关个体判别函数法计算两个体间的全同胞关系。结果 33个Y-STR基因座中有2个基因座存在突变,其中一样本存在19个基因座的缺失。两样本IBS为53,大于阈值42;累积全同胞关系指数为1.36×10~(16),远远大于19;全同胞-无关个体判别函数D_(FS2)D_(R2)。因此倾向于认为两个体为全同胞。结论对于Y染色体微缺失和突变需要进行父系鉴定的情况,可以综合应用IBS法、ITO法以及全同胞-无关个体判别函数法以得出更为可靠的鉴定意见。     

男性Y染色体Amelogenin Y、DYS570及DYS576缺失1例

1 案 例 1.1 简要案情 某水域发现1具高度腐败未知名尸体,法医初步判断死者为男性,提取死者肋软骨进行DNA检测. 1.2 检验方法 使用AutoMate ExpressTM核酸提取系统(美国Thermo Fisher Scientific公司)及配套试剂进行DNA提取,使用PowerPlex? 21系统(美国Pr...     

武汉地区汉族人群线粒体DNA Region V区9bp片段缺失多态性

目的 研究武汉地区汉族群体线粒体DNA RegionV区9bp片段缺失多态性.方法 PCR扩增后采用银染技术分离片段,检测武汉地区汉族群体线粒体DNA RegionV区9bp片段缺失的频率.结果 在武汉地区汉族239个无关个体中发现标准型、缺失型两种多态类型,9bp片段缺失频率为17.15％.结论 武汉地区汉族群体在DNA RegionV区9bp片段存在缺失多态性.     

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。     

Y染色体OSU49基因座遗传多态性分析

目的调查Y染色体OSU49基因座在河南汉族群体中的遗传多态性,评价其法医学应用参数。方法知情同意情况下,采集300名河南汉族男性个体的血样本,荧光标记PCR,扩增产物采用ABI 3130遗传分析仪检测。根据分型检测结果,对不同等位基因进行序列分析(测序)。结果 OSU49基因座包含五核苷酸和四核苷酸两种核心序列。在河南汉族群体中基序表现为:(CTTTC)pCTT(CCCT)7T(CTTTC)1(TCTT)5(TCCT)m(TCTT)n TCT(TCCT)4,五核苷酸核心序列的重复次数为12~17,四核苷酸核心序列的重复次数为20~30,按其片段长度命名等位基因,共发现34个等位基因,GD值为0.918 6,DP值为0.915 5。结论 Y染色体OSU49基因座序列结构复杂,在河南汉族群体中具有较高的遗传多态性,可应用于法医学和人类遗传学研究中。     

孔姓人群Y染色体遗传多态性研究及其法医学意义

目的获取孔姓人群Y-SNP和Y-STR遗传标记的信息,探索姓氏与Y染色体的关联性及法医学意义。方法采用等级复合扩增和SNaPshot技术检测255例孔姓男性和330例随机无关男性样本的12个Y-SNPs位点信息;采用Y filer试剂盒检测孔姓人群的17个Y-STRs基因座;应用Arlequin 3.5.1.2、Network4.6.1.1进行统计分析。结果12个Y-SNPs位点构成13种单倍群分型,孔姓人群和无关人群中最高分布频率的单倍群均为O3a2c1a-M117(21.57%,14.85%)。孔姓人群17个Y-STRs基因座构成的196种单倍型,多态性为0.993 9,单倍型14-12-25-28-19-15-12-19-12-11-12-22-12-11-14-10-19出现15次。O3-M122单倍群的中介网络树及不配对差异分析显示孔姓人群有两个中心星型分布,核苷酸不配对曲线呈单峰泊松分布。结论联合Y-SNP和Y-STR遗传标记分析表明孔姓人群存在复杂的基因交流,有多个姓氏起源,且在历史上经历过一定的扩张或持续增长,结合孔姓家谱历史分析其人群结构的遗传差异在法医学方面有潜在的应用价值。     

男性个体Amelogenin基因座Y片段缺失1例

在法医物证检案中,男性个体Amelogenin基因座Y片段未检出的案例时有报道。笔者在检案中遇到1例,现报道如下。     

Y－染色体SNPs及其在法医学中的应用价值

随着人类基因组计划的迅猛发展,已有越来越多的Y染色体SNP位点被发现,在个人识别、家系谱的建立、疾病的预测与诊断方面,Y染色体单核苷酸多态性提供了非常有价值的遗传标记。同样在法医学中也有广阔的应用前景。本文综合介绍了SNP和Y-SNP的一般特性及在法医学中的应用价值。     

47个SNPs复合检测方法的建立及其法医学应用

目的建立47-plexSNPs复合检测方法，评价其在法医学中的应用价值。方法筛选46个常染色体SNPs和1个Y—SNPs，使用2个检测体系分别对47个SNPs进行单管内复合PCR扩增，采用荧光标记单碱基延伸法和毛细管电泳检测技术进行分型检测；并用建立的方法对260份广东地区无关个体血样进行47个SNPs分型。结果建立的47-plex SNPs的复合检测体系灵敏度高，种属特异性好；260名个体所有SNPs均能准确分型，群体内基因型频率分布均符合Hardy—Weinberg平衡，累积个人识别率大于0．9999，累积非父排除率为0．99982，累积偶合率为6．24×10一。结论本文47-plex SNPs复合检测方法能同时对47个SNPs进行快速、准确的检测，在法医学个体识别鉴定中具有良好的应用前景。     

DNA芯片分析单核苷酸多态性及其法医学应用

DNA芯片技术作为一门新兴的高科技生物技术,显示了它旺盛的生命力和迅猛的发展势头。单核苷酸多态性(SingleNucleotidePolymorphisms,SNPs)是最常见的人类基因组变异类型。它作为一种有效的人类遗传标记,在疾病相关性研究、药物基因组学、法医学、人类进化和迁移等研究中发挥了重要作用。它同DNA芯片技术结合运用也将在法医检验,尤其是亲子鉴定和个人识别中发挥重要作用。本文主要讨论了DNA芯片和SNPs的特点,以及二者联合运用于法医学的价值。     

个体识别SNPs位点组合筛选与法医学应用价值初探

目的筛选用于包括中国主要民族在内的多个群体个体识别的SNPs位点组合体系。方法以Kidd实验室筛选的86个SNPs位点、欧洲SNPforID组织构建的52-plex SNPs复合检测体系为基础,收集和整理这些位点在HapMap数据库中11个人群的分型数据,计算各位点杂合度和Fst值,筛选杂合度〉0.4,Fst值〈0.06,并在研究人群中处于Hardy-Weinberg和连锁平衡的位点组合。针对这些位点,采用MassARRAY分子阵列技术对自行收集的8个人群（尼日利亚人、坦桑尼亚查加人、印度人、丹麦人、俄罗斯汉特人、中国汉族、藏族、维吾尔族）308份样本进行分型,统计群体遗传学参数。结果按本文标准共筛选出66个SNPs位点,均符合Hardy-Weinberg平衡,之间互不连锁,平均杂合度和Fst值分别为0.475、0.014。在本文收集的8个人群中的随机匹配概率在1.45E-24~4.72E-27之间,累积非父排除率为0.999 995 608~0.999 997 876之间。结论本文筛选的SNPs组合系统具有较强的个体识别能力,可用于本文调查的HapMap数据库中11个人群和本文收集的8个人群的个体识别鉴定。     

Measuring the impact of forensic science in detecting burglary and autocrime offences

As UK investment in forensic science has increased, the government has taken a fresh interest in how far this has led to dividends in terms of the detection of crime and its reduction. The Home Office funded 'Pathfinder Project' sought to monitor and document the complex relationships between the collection and use of forensic material (looking at a range of forensic science techniques) and its impact on crime detection. The project specifically targeted the 'volume' crimes of burglary and vehicle crime. Detailed data was gathered on all stages of the process between the collection and use of forensic material and crime detection. The model falls into two conceptual phases--scene attendance to suspect identification and identification to detection. From the analysis it was found that approximately one third of burglary and autocrime scenes are visited by SOCOs. While scientific identifications are only made in a minority of burglary and autocrime offences overall, it belies their importance. About one in ten of burglary and autocrime cases are cleared up by the police and it is estimated that fingerprints and SGMPlus were a contributory factor in achieving one third of these clear ups.     

Establishing an integrated pipeline for automatic and efficient detection of trace DNA encountered in forensic applications

The analysis of trace DNA is a crucial component in forensic applications. Biological materials containing low-level DNA collected at crime scenes, such as fingerprints, can be valuable as evidence. Automatic detection of biological samples has been largely embraced in forensic applications to meet the increasing throughput requirements. However, the amount of DNA automatically retrieved from trace evidence often tends to be small and unstable, ultimately resulting in poor detection of DNA profiles. Thus, in this work, we introduced a robust DNA extraction and purification platform named Bionewtech® BN3200 (Bionewtech®, Shanghai, China) with the goal of constructing a rapid automatic detection system for trace DNA. The establishment of automatic detection system for trace DNA mainly encompassed two parts: assessing the sensitivity of automatic extraction platform and screening the optimal short tandem repeat (STR) typing kit. The sensitivity of Bionewtech® BN3200 platform based on Ultra-sensitive DNA Extraction kit was initially estimated, demonstrating that this extraction platform might contain large potential in the trace DNA extraction. For the amplification part, three sets of commercial multiplex STR typing kits were selected as candidates, and the amplified products were further genotyped on the Applied Biosystems 3500xl Genetic Analyzer. After comparation, SiFa™ 23 Plex Kit was determined as the most suitable amplification system for trace DNA. Eventually, the newly exploited trace DNA detection system was successfully implemented in the detection of fingerprints derived from glass surfaces with the five-seconds contact time. As a result, the DNA recovered from the fingerprints fluctuated approximately from 57.60 pg to 18.05 ng, in addition, over 70% of the total STR loci were detected in 75% of the fingerprint samples.     

Accumulation of population polymorphisms in the mitonuclear genome with probable adaptive effect to extreme environments as altitude for forensic purposes

We analyzed the accumulation of population polymorphism in 2504 individuals - nuclear genomes (nDNA) of 26 populations (81 genes associated to extreme environments) and 3295 mitochondrial genomes (mtDNA) of 47 populations with the aim to found mitonuclear relationship associated an extremes environment as altitude. For that, we use an algorithm developed by us to determine the accumulation of polymorphisms by segments in the genome and thus be able to perform the multivariate analysis to found SNPs differences and similarities among populations. The results showed in Peruvian population a statistically significant mitonuclear relationship for 113/293970 nDNA SNPs in 16/81 genes. In the case of the mtDNA, we found a statistically significant mitonuclear relationship for 6/22 mtDNA positions – Gene. Additionally for the Peruvian population, the MRPP3 had the greatest polymorphism contribution with respect to other populations. Then, these nDNA and mtDNA SNPs in genetically close populations to Peru can be applied to forensic genomic phenotyping to identify groups likely adapted to extreme conditions (such as altitude) or make individualization between low and high altitude populations.     

Genetic diversity and phylogenetic analysis of Chinese Han and Li ethnic populations from Hainan Island by 30 autosomal insertion/deletion polymorphisms

With the characteristics of low mutation rate, length variation and short amplicon size, insertion/deletion polymorphisms (InDels) have the advantages of both short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). Herein, people of two ethnicities from Hainan Island were genotyped for the first time using the Investigator DIPplex kit. We investigated the forensic parameters of the 30 InDels and the phylogenetic relationships among different populations. The accumulated powers of discrimination and powers of exclusion were 0.999 999 999 9646 and 0.9897 in the Hainan Han population and 0.999 999 999 9292 and 0.9861 in the Hainan Li population, respectively. Additionally, population comparisons among geographically, ethnically and linguistically diverse populations via cluster heatmap, multidimensional scaling, principal component analysis, phylogenetic tree and STRUCTURE analyses demonstrated that the Hainan Han population had genetic similarities to the other Han, She and Tujia populations, while the Hainan Li population had close genetic relationships to the Zhuang and Miao groups; both populations had a high degree of genetic differentiation from most Turkic-speaking populations. Aforementioned results suggested that the 30 autosomal InDels are highly polymorphic and informative, which are suitable for human identification and population genetics.