D1S549基因座分型标准物的克隆制备方法及其群体遗传学研究

目的 采用分子克隆技术建立制备大量优质标准 STR基因座等位基因分型标准物的方法,解决长期困扰 STR分型上存在的准确性和标准化问题。方法 先用 PCR扩增出 D1S549基因座的 8个等位基因片段,将其插入 pUC重组质粒中,经 DNA测序分析证实插入片段的结构及大小,用国际标准将插入的等位基因片段进行命名,最后经转染、扩大培养,扩增及再鉴定后制备出标准的 D1S549等位基因分型标准物。结果应用此分型标准物,调查了成都地区汉族 ,甘肃回族及新疆维族群体 D1S549基因座的基因型分布频率。结论 表明该法制备的 STR基因座等位基因分型标准物适用于法医学鉴定实践, D1S549基因座是一个适合我国群体分析和法医学遗传分析的遗传标记。     

用分子克隆技术构建D12S375基因座等位基因分型标准物及汉、回、维族群体遗传学研究

采用分子克隆技术大量制备STR基因座等位基因分型标准物 ,解决长期困扰STR分型的准确性和标准化问题。PCR扩增出D12S375基因座的 7个等位基因片段 ,将其插入pUC重组质粒中 ,经DNA测序分析证实插入片段的结构及大小 ,按重复单位的重复次数对插入的等位基因片段进行命名 ,最后经转染、扩增及再鉴定后制备出标准D12S375基因座等位基因分型标准物。应用此分型标准物 ,调查D12S375基因座在成都汉族 ,甘肃回族及新疆维族群体中的基因型分布频率 ,评估其在法医学实践中的应用价值。     

荧光标记STR复合扩增检验系统的研究

本研究以化学合成的荧光标记引物建立了STR复合扩增体系,体系I包括4个STR基因座和牙釉蛋白基因,体系Ⅱ包括4个STR基因座,体系Ⅲ包括3个STR基因座.制备了体系I各基因座的等位基因标准对照,测定了各等位基因长度.对代表性等位基因进行了序列测定,确定了等位基因重复单位的重复数目及测量长度与等位基因命名的对应关系,对各基因座等位基因进行了标准命名.建立了复合扩增检验系统等位基因自动分析命名的模板.三个复合扩增检验体系的累积随机匹配概率为8.3×10-3,为遗传学分析建立了实用的检验系统.     

荧光复合扩增DXS6804、DXS6799和DXS7132基因座

应用分子克隆技术制备出DXS6804、DXS6799和DXS7132基因座的等位基因分型标准物,通过研究复合扩增技术,构建3个基因座X—STR复合扩增的荧光检测体系。     

4个Y-STR基因座遗传多态性及分子克隆技术制备分型标准物

目的研究Y染色体4个新发现的STR基因座在成都汉族群体中的遗传多态性,寻找适合于法医学应用的Y-STR基因座并用分子克隆法制备其等位基因分型标准物。方法用PCR扩增和非变性聚丙烯酰胺凝胶电泳技术,对105名成都地区汉族无血缘关系男性个体的4个Y-STR基因座进行分型。并通过分子克隆技术制备DYS643基因座的等位基因分型标准物。结果DYS632、DYS634、DYS642和DYS643四个STR基因座均具有Y染色体特异性,在成都汉族群体中等位基因个数分别为2、4、3和5,共检测出31种单倍型;DYS643基因座的等位基因分型标准物可以用于群体研究。结论DYS643基因座及其分子克隆法制备的等位基因分型标准物具有较高的法医学应用价值。     

壮族人群3个STR基因座基因频率分布及其法医学应用

研究3个STR基因座（D21S11、HumFGA、D19S253）在广西壮族人群中的基因频率分布及其在实际检案中的应用价值。以自制等位基因Ladder样品作为标准对照,用PCR结合PAGE技术对3个STR基因座的扩增产物进行分型。结果显示：D21S11基因座有14个等位基因,有44个基因型;HumFGA基因座有15个等位基因,40个基因型;D195253基因座有9个等位基因,23个基因型。经检验,3个STR基因座基因型分布均符合Hardy－Weinberg平衡,累计个体识别力（DP）为0．9995。3个STR基因座在壮族人群属高识别力遗传标记系统,在法医学个体识别及亲权鉴定方面有重要价值。     

基于重组质粒制备STR分型阳性参照物

目的基于重组质粒制备可用于校准法医STR分型的阳性参照物。方法以常用阳性参照物9948人类基因组DNA STR分型为依据,基于重组质粒构建包含CSF1PO、D7S820、TH01等40个常染色体位点,DYS391、DYS522、DYS385a/b等22个Y染色体位点以及性别判定基因座Amelogenin的STR分型阳性参照物。将重组质粒定量、稀释后等比例混合,分别应用于DNATyper~?19、DNATyper~?24、DNATyper~?Y、Amp F?STR~?Identifiler~?Plus以及Power Plex~?18D System五种扩增试剂盒。结果阳性参照物中各重组质粒浓度为0.01pg/μL~0.001pg/μL;应用于Amp F?STR~?Identifiler~?Plus PCR扩增试剂盒,基于重组质粒制备的阳性参照物与人类基因组DNA扩增检测结果差异较小;将此阳性参照物分别应用于不同公司、不同STR基因座的四种STR扩增试剂盒,电泳检测图谱显示各基因座基因型完整,分型正确,峰高相当,基因座间均衡性良好。结论基于重组质粒制备STR分型阳性参照物,是一种可以替代细胞系制备阳性参照物的方法,具有一定的参考价值。基于此方法制备的阳性参照物可适用于市面上常用的STR检验试剂盒,普适性较强,对法医DNA分型检测有一定的实用价值。     

等位基因分型标准物的法医应用及制备研究

短串联重复序列(shorttandemrepeats,简称STR)由于其本身的优点和检测技术的不断完善,在今后相当长的时间内仍将是法医应用的重要遗传标记。等位基因分型标准物是STR检测试剂盒的组成部分,能够保证各等位基因分型的准确性。本文对等位基因分型标准物的出现、在法庭科学上的应用以及目前制备方法进行了综述,并对各种制备方法进行比较,以期对各实验室按照实际需要自行制备相关STR的等位基因分型标准物在方法选择上有所帮助,从而更好地进行法医物证鉴定。     

STR图谱分析中的问题及对策

STR图谱分析是STR检验的最后一步,对STR图谱正确分析解读会影响并决定数据的有效性。在STR图谱分型中常见的影响因素有以下几种：出现OL峰,其常见原因有稀有等位基因、电泳漂移、pullup峰、染料峰、荧光污染峰等;出现微量DNA致等位基因丢失,需要多次扩增或浓缩后扩增来帮助判断等位基因的个数;出现引物结合区的变异致无效等位基因,可以通过不同试剂盒的扩增来纠正[1];出现三等位基因,常见的原因有疾病或体细胞突变,疾病会致染色体和基因的复制影响到个体的所有细胞,     

等位基因分型标准物的法医应用及制备研究

短串联重复序列（short tandem repeats，简称STR）由于其本身的优点和检测技术的不断完善，在今后相当长的时间内仍将是法医应用的重要遗传标记。等位基因分型标准物是STR检测试剂盒的组成部分，能够保证各等位基因分型的准确性。本文对等位基因分型标准物的出现、在法庭科学上的应用以及目前制备方法进行了综述，并对各种制备方法进行比较，以期对各实验室按照实际需要自行制备相关STR的等位基因分型标准物在方法选择上有所帮助，从而更好地进行法医物证鉴定。     

D11S4463基因座等位基因分型标准物制备及应用

目的制备D11S4463基因座等位基因分型标准物,并调查该基因座在中国汉族人群中的遗传多态性。方法设计引物,用荧光引物PCR扩增和ABI 3130XL遗传分析仪电泳的方法,对中国汉族520份无关个体血斑样本D11S4463基因座遗传多态性进行调查,用分子克隆的方法构建其等位基因分型标准物。结果 D11S4463基因座在中国汉族人群中共检测出9个等位基因,杂合度、匹配概率、个体识别能力、多态性信息含量及非父排除概率分别为0.735、0.089、0.911、0.73、0.485。应用分子克隆的方法成功构建其等位基因分型标准物,按国际法庭血液遗传学学会推荐的原则命名。结论 D11S4463基因座具有较高的遗传多态性,应用分子克隆的方法构建其等位基因分型标准物,在法医科学实践中具有较高的应用价值。     

The different mobility of complementary strands depends on the proportion AC/GT

The electrophoretic mobility of DNA fragments on denaturing polyacrylamide gel depends on various factors. One of these is the base composition of a single-stranded DNA (ssDNA). We confirmed that one strand and its complementary strand of polymerase chain reaction (PCR) products migrated with different mobilities in all alleles detected at 12 out of the 13 short tandem repeat (STR) loci studied. The mobility differences between complementary strands (MD) were also observed regardless of end-polishing with Pfu DNA polymerase. MD was therefore not influenced by additional nucleotides to each strand of the PCR products. We then analyzed the relation between MD and the base composition using one representative allele at each of the 13 loci. The results indicated that MD was affected by the adenine plus cytosine (AC) content in the ssDNA and was proportional to the values of the AC content divided by the guanine plus thymine (GT) content in the AC-rich strand (the proportion AC/GT). When the proportion AC/GT was well-balanced, MD decreased. The same tendency was observed even in the end-polished strands. In this study, the electrophoretic mobility of an ssDNA on denaturing polyacrylamide gels was shown to depend on the proportion AC/GT. Unless the same side of the PCR products is labelled in the context of a PCR-based STR typing, distinct alleles may be mistaken for identical ones because of the different mobility of complementary strands. Accordingly, the labelled strand should be described if only one strand of the PCR products is detected. When using an allelic ladder marker as a size standard, the labelled side should be unified between STR alleles and the allelic ladder alleles.     

Sequence determination of an allelic ladder for the STR polymorphism at the CD4 locus and application of the ladder in testing an Austrian Caucasian population sample

The short tandem repeat (STR) polymorphism at the CD4 locus, designated HUMCD4. was examined by PCR, native polyacrylamide electrophoresis and subsequent silver staining using an allelic ladder of eight distinguishable alleles occurring in an Austrian Caucasian population sample as a standard size marker. The ladder was produced by pooling equal concentrations of eluted, separately amplified and sequenced alleles, which were previously identified by their different electrophoretical migration. Components of the ladder are in regular intervals of five basepairs. Alleles 4 to 8 were designated according to the number of AAAAG repeat units. The four longer alleles 8′ to 11 showed a stable A to G transition in one of the repeat units and were designated counting the AAAGG unit for a AAAAG. Allele 8′ was not included in the ladder because it showed the same electrophoretic mobility as allele 8. This ladder proved to be a precise and reliable tool in the analysis of 600 chromosomes of the Austrian population. The population investigated showed no deviation from Hardy-Weinberg equilibrium (P = 0.23).     

分子克隆技术制备miniSTR基因座等位基因分型标准物

目的调查D9S2157、D9S1122、D10S1435、D12ATA63和D2S1776共5个miniSTR基因座在河北汉族人群中的遗传多态性,并用分子克隆的方法制备等位基因分型标准物。方法用荧光PCR和ABI 310遗传分析仪对河北汉族120份无关个体血样中的5个miniSTR基因座进行遗传多态性分析。用分子克隆的方法构建等位基因分型标准物。结果5个基因座在河北汉族人群中分别检测出了8、8、7、5和8个等位基因,多态信息含量分别为0．790、0．720、0．750、0．630和0．850。结论5个基因座在河北汉族人群中有较高的多态信息含量,其分子克隆法制备的等位基因分型标准物具有较高的法医学应用价值。     

D18S872基因座在汉、维、蒙古、回族群体中的遗传多态性研究及其应用

目的　调查D18S872基因座在成都汉族 ,新疆维族和蒙古族 ,甘肃回族 4个民族中的遗传多态性 ,获得群体遗传学基本数据。　方法　等位基因分型标准物制备采用分子克隆技术 ,样本基因分型采用PCR和PAG垂直电泳技术、银染显色方法。　结果　获得D18S872基因座等位基因分型标准物及该基因座在 4个群体中的遗传学数据。　结论　结果表明D18S872基因座在法医学个人识别和亲子鉴定中有一定的应用价值。     

Robustness of the Y STRs DYS19, DYS389 I and II, DYS390 and DYS393: optimization of a PCR pentaplex

Various technical methods were investigated with the aim of developing a multiplex system to amplify five Y-chromosome STR loci in the same PCR reaction: DYS393, DYS19, DYS390, DYS389 I and DYS389 II. A sequenced allelic ladder was constructed with previously sequenced alleles including the most common ones. A number of reamplification conditions of the allelic ladders were tested. The pentaplex was evaluated for typing using two different platforms (ABI and ALF) with promising results. However, in degraded samples non-specific artifacts were observed in the DYS393 system in the same range of sizes as the real alleles. This system can also be typed in females under relatively low stringency conditions in the PCR amplification, making this system prone to errors in critical samples. This lack of specificity can be reduced by increasing the stringency of the PCR conditions. The DYS19 ladder cannot be reamplified as stutters appear after a few reamplifications. These stutters are probably due to a 2 bp slippage induced by the presence of a TA repeat stretch in the PCR amplified fragments. Non-specific products were also noted in the DYS389 I and DYS389 II amplification, although out of the range of other alleles in this pentaplex. This newly constructed pentaplex has proved to be very useful in population genetic studies because all five Y STR markers can be loaded in the same lane of a gel with other Y STR singleplex or multiplexes. The usefulness of Y-chromosome STRs in criminal casework is especially evident in analyzing azoospermic individuals.