应用MVR-PCR和银染法分析河北汉族人群D1S8基因座DNA结构多态性

研究D1S8基因座重复序列内部的变异,并进行数字编码。应用MVR－PCR和聚丙烯酰胺梯度凝胶电泳银染法对240名河北汉人无关个体进行检测。结果每个个体得到约30个数字编码,未发现任何两无关个体所有编码相同,30个编码完全相同的概率为3．55×10－11。3种重复单位a-型、t-型、o-型出现的频率分别为54.77％、42．54％、2．69％。为小卫星变异重复序列的研究及其在法医实践中的应用提供了一种新方法。     

数字编码小卫星可变重复序列法对中国汉人分型

介绍用辣根过氧化物酶标记的寡核苷酸探针检测的数字编码小卫星MS32可变重复序列方法。经用此法研究发现，中国汉人可得到50个编码以上，无关个体前50个编码中至少有20个编码不同，未发现两个无关个体的编码相同，50个编码全部相同的概率为4．09×10-16。三种重单位，即a-型、t-型和o-型出现比例分别为61．5％、32％和6．5％。MVR－PCR得到的数字编码具有高度的变异性，识别率高，为法医物证检验提供一个新的个人识别方法。     

中国汉族人群2个高变MVR基因座遗传多态性

常染色体小卫星基因座D16S309(MS205)位于16p13.3(AE006466)[1],核心序列为45~54bp,重复次数8~87次[2]。D2S44(YNH24)基因座位于2q21.3-q22(AJ001534)[3],核心序列为31bp。本文采用MVR-PCR技术,选择重复单位3′侧翼区引物(公共引物)和MVR特异性引物匹配进行PCR扩增,对中国河北汉族人群2个基因座遗传多态性进行了调查。1材料与方法1.1样本及DNA提取116份枸橼酸钠抗凝血样由河北省血液中心提供,采自无血缘关系汉族健康个体。常规饱和酚-氯仿法提取DNA。1.2MVR-PCRD16S309引物序列参见文献[2,4]。反应体系为25μl,分别加入5μl…     

HRP标记探针MVR分型法在法医学上的应用

采用辣根酶标记寡核管酸探针数字编码小卫星MS可变重复序列（HRP标记探针MVR方法）,检验微量生物材料。结果表明,相当于5μl血液的血痕、单根有毛囊的毛发以及相当于4μl唾液的斑迹均获得了清晰易读的MVR图谱,灵敏度达1ng。将此方法应用于检案实践取得了较好的效果。MVR－PCR技术在法医学个人识别和亲子鉴定方面极有应用前景。     

MS31A位点MVR-PCR技术在法医学鉴定中的应用

应用小卫星特异性引物31A、31A -A、31A -G与α -32PdCTP掺入扩增技术对MS31A(位于D7S21位点)进行MVR -PCR分析 ,成功地建立了一套适合于刑事案件中微量血痕、混合斑、毛发、骨组织等样品鉴定的快速、简便、准确的分析技术。灵敏度分析表明 ,该方法能检测出低至1ng的基因组DNA ,具有较高的灵敏性。本技术在40余起强奸、凶杀等疑难案件中应用均获得成功。本研究还利用MVR数据库资料对实际案件中的犯罪嫌疑人成功地进行了筛选、排查以及串并案工作。     

STR联合扩增鉴定碎尸案1例

聚合酶链反应（PCR）技术于1985年由美国Cetus公司的RandallSaiki、HenryErlich和KaryMullis等联合创建[1]。人类基因组DNA有3×109bp，其中10％是串联重复序列，被称为卫星DNA。卫星DNA按重复单位的长短分为大卫星、中卫星、小卫星和微卫星。其中微卫星重复单位仅由2～7hp组成，故又称为短串联重复序列（STR）。Saihi等人发现人类STR可用PCR方法扩增[2]，并且有高度多态性[3]。作者成功地利用PCR扩增PLA2A、VWA、CYP19、TH01、LPL、D6S366、D19S253、FESFPS八个STR位点对一例碎尸案中尸块做同一认定及尸源鉴定，现…     

MVR-PCR分析技术的研究进展

<正> 小卫星DNA通常是指以10～50bp为一重复单位,即核心序列(Core Sequence)存在于基因组非编码区的串联重复序列。小卫星DNA核心序列变异(Minisatellite Variant Ropeat,MVR),不仅存在核心序列串联数目的变异,而且核心序列的组成亦会有微小变异。     

p33.4位点扩增片段长度多态性及其法医学应用的研究

以聚合酶链反应（PCR）、聚丙烯酰胺凝胶垂直电泳和银染法对中国100名无关个体小卫星区域p33．4位点的扩增片段长度多态性（Amp－FLPs）进行了研究，检出了8个等位基因。通过BIOTRAC系统进行数据处理．各等位基因重复单位的数目分别为7、10到15，其中在13～14之间发现一差值不足一个重复单位长度的罕见等位基因。片段长度分布于603～1115bP之间，基因频率分布于0.5～33．5％间，杂合度为64％，DP值为84．5％。对5个家庭25名相关个体进行分析，符合孟德尔遗传定律；对同一个体不同组织的DNA进行P33.4位点的分型研究表明，该技术适宜于法医物证检验。此外，本研究以Chelex处理不同检材制备DNA模板用于扩增，率先建立了比常规方法更快、更简便、更为实用的检验方法。     

PCR技术在法医学鉴定中的应用

近年来国内法医研究人员已先后将DNA遗传指纹图技术、单位点VNTR以及PCR－VNTR、PCR－ASO、PCR－STR、MVR－PCR。PCR．SSCP’等技术应用于法医学鉴定，使法庭科学对犯罪生物物证的个人识别能力的研究有了突破性进展，取得了令人瞩目的成就。笔者根据现有实验室条件和实际办案工作的需要，对DIS80、ApOB、HUMACTBPZ、HUMTH01等位点进行分析研究，除进行必要的基因频率调查、家系遗传性分析外，通过对天津地区60余起案件的检验，重点研究实际检案中的问题。材料和方法一、实验主要仪器、试剂卫．DNA热循环仪：（PCT…     

HLA-A位点DNA分型及其法医学应用

应用聚合酶链反应0寡核苷酸探针（PCR－SSO）斑点杂交技术，对222名辽宁地区汉族人群无关个体进行HLA－A基因检测，研究中国辽宁地区汉族群体的HLA－A座位基因分布状况。共检出HLA－A等位基因24个，其中以等位基因HLA－A0201最为常见，频率为0．2635；依次是2402101和1101，等位基因频率分别为0．1847和0．1262.理论杂合度为87％，个人鉴别机率为92％，非父排除率为73．3％。在中国辽宁汉族中检出73种基因型，对观察值和期望值进行X2检验，符合Hardy－Weinberg平衡定律（x2＝6．28，df＝9，0．5＜P＜0．75）。家系分析结果表明按照孟德尔方式遗传。提出的中国辽宁汉族HLA－A等位基因的遗传基因情况，可用于法医学个人识别和亲子鉴定。人类学，HLA相关疾病，及器官移植研究。     

The potential contribution of MVR-PCR to paternity probabilities in a case lacking a mother.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was applied to a paternity case lacking a mother to evaluate the paternity probability. After three flanking polymorphic sites at each of MS31A and MS32 loci were investigated from the child and alleged father, allele-specific MVR-PCR was performed using genomic DNA. It was confirmed that one allele in the child was identical to that in the alleged father at both loci. Mapped allele codes were compared with allele structures established from population surveys. No perfect matches were found although some motifs were shared with other Japanese alleles. The paternity index and probability of paternity exclusion at these two MVR loci were then estimated, establishing the power of MVR-PCR even in paternity cases lacking a mother.     

中国汉族与日本群体DYFl55S1基因座的遗传多态性

目的探讨Y染色体DYF155S1基因座的遗传多态性及群体间差异。方法 应用MVR-PCR、荧光显谱及DNA序列分析技术,对来自中国群体(北方汉族,64例)和日本群体(43例)男性个体的DYF155S1基因座进行初步分析。结果107例样本共检出了5种重复序列类型,包括新的命名为6型的重复序列,它是在1型的基础上T22A置换所形成,仅存在于日本群体,可作为民族特征性遗传标记。2群体重复序列的排列方式以3134顺序为主,在中国和日本群体中各占73.44％和67.44％,是黄种人的特点。134顺序在中国群体中占第二位,为17.19％,6134排列占日本群体的16.28％。3’端的4型重复序列的平均数目在日本群体为8.8条,明显低于中国群体的12.5条。结论DYF155S1基因座具有非常高的遗传多态性和明显的群体差异。     

Distinguishing minisatellite mutation from non-paternity by MVR-PCR

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was devised to map the interspersion pattern of subtle variant repeats along minisatellite tandem arrays. MVR-PCR has revealed enormous diversity of allele structures at several loci, far more than can be resolved by allele length analysis. We have reported the application of MVR-PCR at minisatellite MS32 (D1S8) and MS31A (D7S21) in a paternity case lacking a mother and showed that it resulted in higher paternity probabilities than for a set of 12 other DNA markers including six STRs. Hypervariable minisatellites like MS32 and MS3lA can however, show significant germline mutation rates to new length alleles which can generate false exclusions in paternity cases although paternity cases showing mutant paternal alleles at more than one locus will be rare when several MVR loci are examined. Detailed knowledge of mutation processes coupled with MVR analysis of allele structure can help distinguish mutation from non-paternity. We now show how similar mutant alleles are to their progenitors using both real and simulated data, and demonstrate how MVR-PCR can be used to identify mutant paternal allele in paternity cases showing apparent exclusions.     

The application of minisatellite variant repeat mapping by PCR (MVR-PCR) in a paternity case showing false exclusion due to STR mutation

A boy and a girl with their mother brought a paternity suit against an alleged but deceased father. We tested six conventional genetic markers, the AmpliType PM+ DQA1 and twelve STR loci the children and mother together with the alleged paternal grandparents. We also DNA typed the bloodstain found later in the alleged father's medical record. Only the result at D3S1358 in a nineplex STR system excluded the alleged father from parentage of the boy, whereas all markers were inclusive for the girl. Accordingly, we performed sequence analysis at D3S1358 to confirm the presence of a paternal exclusion or mutation. The sequence analysis indicated that the boy's allele 17 could have originated from either of the alleged father's allele 16 or 18 by a single-step mutation associated with slippage mutation in STR loci. We carried out minisatellite variant repeat mapping by PCR (MVR-PCR) at loci D1S8 (MS32) and D7S21 (MS31A) and mapped allele haplotypes of all individuals except the deceased alleged father. The MVR-PCR analysis showed that the boy has no inconsistency with the relationship between the alleged grandparents, and was very effective at increasing the paternity index (PI) value. We conclude that there is biological relationship between not only the girl but also the boy and the alleged father.     

Population distribution of six PCR-amplified loci in Madeira Archipelago (Portugal).

Frequency data of the short tandem repeat (STR) loci HUMTH01, HUMVWA31/A, HUMF13A1, HUMFES/FPS, D12S391 and HUMFIBRA/FGA were determined in blood stains obtained from a population of unrelated individuals from the Madeira Archipelago. The observed genotype distribution showed no significant deviation from the Hardy-Weinberg equilibrium and there was no evidence for association of alleles among the six loci. Population data showed a combined discrimination power of 0.9999998 and a chance of exclusion of 0.99597. The frequencies are similar to those of other compared caucasian populations but significant differences were found between the Madeira population and Japanese, Chinese, Greenland Eskimos and Quechua Amerindians. The six loci studied, together proved to be highly discriminating and valuable for forensic cases.     

Forensic application of reverse dot blot with biotin incorporation

A rapid and accurate reverse dot blot technique was successfully established. It could distinguish the 6 alleles of 0101, 0102, 0103, 0201, 0301 and 0401 of HLA-DQA locus. Alleles and genotype frequencies for HLA-DQA locus were determined in 200 unrelated individuals of northern Han population. The discrimination power (DP) was 0.928, heterozygocity (H) 0.75. By direct biotin incorperation, 1 ng genome DNA could be detected successfully. This method can be used for paternity test and individual identification in forensic science practice.     

D19S400基因座在中国3个汉族群体与日本人中的遗传多态性研究

为了解中国广州、吉林、成都三个地区汉族和日本人群体D19540O基因座基因频率分布，并获得中国三个汉族群体和日本群体D19M00基因座的群体遗传数据，比较它们之间的遗传学差异，探究在法医学应用中的意义。应用PCR扩增技术，聚丙烯酸胺凝胶垂直板电泳对D19S400基因座分型。在四个群体469个个体中共检出11个等位基因，45种基因型，基因型频率分布均符合Hardy-Weinberg平衡。各群体的观察杂合度为0.75～0.84，非父排除概率为0.6057～0.6582，个人识别机率为0．9301～0．9480。四个群体之间基因频率分布无显著性差异（P＞0．05）。结果表明，D19S400基因座在群体遗传学研究和法医学个人识别中有较高应用价值。