西藏藏族人群18个STR基因座遗传多态性

<正>DNA Typer 15TMplus试剂盒是公安部物证鉴定中心针对目前办案及DNA数据库建设研制的五色荧光STR复合扩增试剂盒。该试剂盒在DNATyper15TM试剂盒的基础上,增加了4个基因座(TH01、TPOX、D19S433、D12S391)。本文使用该试剂盒,对西藏藏族人群18个STR基因座进行了遗传多态性调查,现报道如下。     

两无关个体单亲亲子鉴定不排除1例

1案例资料2004年4月5日,黄埔分局送来在某医院提取的两名涉嫌被拐卖男童(均约6个月大)的口腔拭子,要求进行DNA检验,以确定是否有血缘关系。用Chelex-100法提取该两名儿童的口腔拭子DNA。先后用Identifiler试剂盒、PowerPlex 16试剂盒、FFFL试剂盒和PowerPlex Y试剂盒进行扩增。扩增产物用AB I 3100型遗传分析仪检测分型,并进行m tDNA L15997~L16401片段序列测定。经Identifiler系统15个常染色体STR基因座检验,两男孩的基因型不同,但每个基因座均至少有1个相同的等位基因,见表1。再使用Power Plex16和FFFL试剂盒检验,在增加的…     

湖南地区汉族人群18个STR基因座遗传多态性

本文应用DNATyper19直扩试剂盒（公安部物证鉴定中心研制）,对湖南地区汉族人群18个STR基因座进行遗传多态性调查,为法医学研究和实践提供基础数据。1材料与方法1.1样本从湖南省违法犯罪人员数据库筛选获得2 437份湖南地区汉族无关个体血样（博坤公司血样采集卡）,样本来源地覆盖全省14个市州。1.2 STR扩增及分型检测采用DNATyper19直扩试剂盒进行直接扩增,     

X-STR分型在特殊二联体亲子鉴定中的应用1例

1 案例 1.1简要案情 两名男子委托本鉴定所对他们与1名女孩是否存在亲生血缘关系进行鉴定. 1.2检验过程 本鉴定所采集3人指血,Chelex-100法提取DNA,采用PowerPlex(R) 21系统(美国Promega公司)检测20个常染色体STR基因座以及1个性别基因座,采用Investigator Argus X-12试剂盒(德国QIAGEN公司)检测12个X-STR基因座,以上扩增均在9700型基因扩增仪(美国AB公司)上完成,扩增体系和条件均按照使用说明书操作,扩增产物在3130遗传分析仪(美国AB公司)上进行毛细管电泳,用GeneMapper ID v3.2软件进行基因分型.     

DNATyper19~(TM)与Identifiler~(TM)Plus试剂盒对疑难检材检验比较

<正>本研究应用DNATyper19~(TM)试剂盒和Identifiler~(TM) Plus试剂盒对骨骼、牙齿和脱落细胞等疑难检材DNA进行平行扩增,从分型结果一致性、检出率等方面进行分析,探讨DNATyper19~(TM)试剂盒在疑难检材检验中应用的可行性[1-5]。1材料与方法1.1样本来源510例生物检材均来自本实验室日常检案积累,其中包括:骨骼70例、牙齿50例、脱落细胞390例(包     

同母异父姐妹10个X-STR有相同等位基因1例

1案例资料 1.1样本及检验方法 被鉴定人为两位女士（S1和S2）及其父亲（F2）,要求鉴定两位女士是否为姐妹关系。X-STR分型按文献[1]方法进行,常染色体STR分型使用PowerPlex 16 System试剂盒。用软件Merlin[2]计算同胞或半同胞指数。     

19例基因突变分析

目的对19例不符合遗传规律的基因突变现象进行分析，并探讨其对亲子鉴定的影响。方法对使用Profiler Plus＋Cofiler试剂盒鉴定的118例，使用Identifiler^TM试剂盒鉴定的552例亲子鉴定案例中出现的19例1～2个STR不符合遗传规律的家庭，使用DNATyper15^TM试剂盒进行复检。结果原鉴定19个案例中有1个STR基因座不符合遗传规律的17例，有2个STR基因座不符合遗传规律的2例；使用DNATyper15^TM试剂盒进行复检，结果其中3例D8S1179基因座被证实系等位基因丢失，其余16例复检结果仍不符合遗传规律，分析认为系等位基因突变所致。结论在有1～2个基因座不符合遗传规律时，要综合分析，并增加检测基因座的数量，避免基因座的错误分型和亲子关系的误判。     

15个常染色体和10个Y-STR基因座复合扩增试剂盒的研发

目的建立法医物证学常染色体和Y染色体综合检测的方法。方法选择常用的常染色体STR基因座和Y染色体STR基因座,设计复合扩增引物,形成五色荧光标记复合扩增试剂盒。结果开发出一个五色荧光标记复合扩增试剂盒,可同时对15个常染色体STR基因座、1个性别基因座和10个Y染色体STR基因座进行分型检测。结论 15个常染色体STR加10个Y染色体STR检测试剂盒结合毛细管电泳凝胶进行STR分型,结果准确可靠,在法医学案件检验及数据库建设等方面有良好的应用前景。     

采用STR和SNP遗传标记鉴定全同胞姐妹关系

目的 通过对常染色体和X染色体遗传标记的检测,探讨全同胞姐妹关系的鉴定策略.方法 提取姐妹个体的DNA,采用SinofileTM试剂盒检验常染色体上的15个STR基因座、采用Mentype(R) Argus X-8试剂盒和多重X染色体STR检测试剂盒检验X染色体上的17个STR基因座,同时采用TaqMan技术对11个X-SNP位点进行分型检测.结果 依据常染色体STR基因座的检测结果计算全同胞指数,不排除被检个体的同胞姐妹关系:X染色体上各个STR基因座和SNP位点均检见1～2个相同的等位基因,进一步支持被检个体的同胞姐妹关系.结论 对于全同胞姐妹关系的鉴定案例,除了检测常染色体STR基因座外,还可以从X染色体上选择多态性遗传标记进行检测,获得更多的遗传信息.     

中国汉族人群21个常染色体STR的遗传多态性

本文对中国汉族608个无关个体的常染色体STR基因座遗传多态性进行调查。结果 21个常染色体STR基因座检出的频率分布在中国汉族人群中均符合Hardy-Weinberg平衡。Globalfiler~(TM)试剂盒所包含的STR基因座具有良好的遗传多态性,对法医学应用和群体遗传学研究具有重要意义。     

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。     

Large-scale concordance study for 16 autosomal STRs analyzed with PowerPlex ESI17 and NGM SElect

This study aimed at identifying the frequency of kit-dependent discordances and genetic specialties in STR DNA typing, which is particularly important for the interpretation of DNA profiles as well as national and international database searches. We analyzed a total of 27′510 buccal swabs with the PowerPlex ESI 17 and NGM SElect amplification kits and documented discordances and other anomalies. We found kit-dependent full dropouts to be the most frequent events, most of which were associated with the NGM SElect kit. The total kit-dependent discordance rate determined in this study amounts to 0.74%. With this dataset, we also provide dropout rates, which can be used to estimate silent allele frequencies for 16 common STR loci amplified with the PowerPlex ESI17 and NGM SElect amplification kits.     

PowerPlex 21与GoldeneyeTM 20A试剂盒的一致性验证及法医学应用

目的：确认PowerPlex 21试剂盒与GoldeneyeTM 20A试剂盒分型结果的一致性。方法应用两试剂盒对205名北京汉族无关个体血样DNA进行复合扩增,观察19个重叠STR基因座分型的一致性,并统计D1S1656的遗传多态性。结果所有19个重叠基因座分型相同,两个试剂盒的杂合基因座峰高比例差异无统计学意义（P〉0.05）。D1S1656杂合度为0.878,个人识别率为0.949,三联体非父排除率为0.751,二联体非父排除率为0.506,多态信息含量为0.810。结论 PowerPlex21试剂盒与GoldeneyeTM 20A试剂盒分型结果一致性好,引物设计合理;D1S1656多态性好,可用于人类遗传分析及法医学中的亲子鉴定和个人识别。     

Validation of a male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.     

Validation of male-specific, 12-locus fluorescent short tandem repeat (STR) multiplex

Y chromosome-specific short tandem repeat (Y-STR) analysis has become another widely accepted tool for human identification. The PowerPlex Y System is a fluorescent multiplex that includes the 12 loci: DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. This panel of markers incorporates the 9-locus European minimal haplotype (EMH) loci recommended by the International Y-STR User Group and the 11-locus set recommended by the Scientific Working Group on DNA Analysis Methods (SWGDAM). Described here are inter-laboratory results from 17 developmental validation studies of the PowerPlex Y System and include the following results: (a) samples distributed between laboratories and commercial standards produced expected and reproducible haplotypes; (b) use of common amplification and detection instruments were successfully demonstrated; (c) full profiles were obtained with standard 30 and 32 cycle amplification protocols and cycle number (24-28 cycles) could be modified to match different substrates (such as direct amplification of FTA paper); (d) complete profiles were observed with reaction volumes from 6.25 to 50 microL; (e) minimal impact was observed with variation of enzyme concentration; (f) full haplotypes were observed with 0.5-2x primer concentrations; however, relative yield between loci varied with concentration; (g) reduction of magnesium to 1mM (1.5 mM standard) resulted in minimal amplification, while only partial loss of yield was observed with 1.25 mM magnesium; (h) decreasing the annealing temperature by 2-4 degrees C did not generate artifacts or locus dropout and most laboratories observed full amplification with the annealing temperature increased by 2 degrees C and significant locus dropout with a 4 degrees C increase in annealing temperature; (i) amplification of individual loci with primers used in the multiplex produced the same alleles as observed with the multiplex amplification; (j) all laboratories observed full amplification with >or = 125 pg of male template with partial and/or complete profiles observed using 30-62.5 pg of DNA; (k) analysis of < or = 500 ng of female DNA did not yield amplification products; (l) the minor male component of a male/female mixture was observed with < or =1200-fold excess female DNA with the majority of alleles still observed with 10,000-fold excess female; (m) male/male mixtures produced full profiles from the minor contributor with 10-20-fold excess of the major contributor; (n) average stutter for each locus; (o) precision of sizing were determined; (p) human-specificity studies displayed amplification products only with some primate samples; and (q) reanalysis of 102 non-probative casework samples from 65 cases produced results consistent with original findings and in some instances additional identification of a minor male contributor to a male/female mixture was obtained. In general, the PowerPlex Y System was shown to have the sensitivity, specificity and reliability required for forensic DNA analysis.     

Validation and implementation of the PowerPlex 16 BIO System STR multiplex for forensic casework

The PowerPlex 16 BIO multiplex short tandem repeat (STR) system contains the 13 CODIS loci (FGA, TPOX, D8S1179, vWA, D18S51, D21S11, TH01, D3S1358, CSF1PO, D16S539, D7S820, D13S317, and DS5S818), plus two pentanucleotide repeat loci (Penta D and Penta E) and the sex-identifying locus. Amelogenin. The PowerPlex 16 BIO System is optimized for use with the Hitachi FMBIO gel imaging systems. A consortium of seven independent laboratories collaborated to perform the studies defined by the FBI standards for performing a developmental validation, including the evaluation of sample concordance, percent stutter determination, nonprobative casework, precision, sensitivity, mixture determination, effect of substrates, the impact of environmental insults, and species specificity. All samples tested for concordance were consistent except for one sample from the Virginia Division of Forensic Science database that displayed discordance at D13S317, a locus whose primer sequence was altered. Stutter values were comparable to those of other STR multiplex systems, the precision was comparable to other multiplexes analyzed by gel electrophoresis, the DNA profiles were unchanged by the substrate upon which the blood samples were placed, and the nonprobative casework samples re-typed for the PowerPlex 16 BIO System were consistent with previous typing results. When greater than 0.125 ng of DNA was placed into the PowerPlex 16 BIO System amplification reaction, a full profile was generated by all laboratories. The mixture study results were comparable to those reported for other multiplex systems, the environmental study demonstrated a loss of larger molecular weight loci when samples were incubated at elevated temperatures for a prolonged period of time, and the only notable cross species hybridization was observed with primate DNA samples. This extensive validation work performed demonstrates that the PowerPlex 16 BIO System provides STR data of a quality comparable with other PowerPlex STR multiplex kits as well as other widely used STR multiplexes and is thus suitable for evidentiary casework analysis as well as database sample profiling.     

Uses of the NIST 26plex STR assay for human identity testing

Ongoing work at the U.S. National Institute of Standards and Technology has focused on the characterization of 26 autosomal STR loci for human identity testing. These 26 loci are in addition to the existing 13 U.S. core loci and those found in PowerPlex16 and Identifiler commercial STR typing kits. The amplification of the 26 loci has been optimized for degraded extracts in unique miniplex panels and also for reference samples as a single reaction 26plex assay. A study has been performed comparing genotypes obtained with the 26plex primers to those with miniplex panels for allele drop out and concordance. The forensic utility of the 26plex assay was evaluated for situations where additional loci are beneficial. The utility of this large multiplex was also tested in a case involving DNA extracted from degraded bone samples. The 26plex can serve as a low-cost assay (compared to commercially available kits) useful for both sorting comingled remains and providing additional markers for increased statistical support for samples that require “non-trio” family references for human identification.     

An updated validation of Promega's PowerPlex 16 System: high throughput databasing under reduced PCR volume conditions on Applied Biosystem's 96 capillary 3730xl DNA Analyzer

The PowerPlex 16 System from Promega Corporation allows single tube multiplex amplification of sixteen short tandem repeat (STR) loci including all 13 core combined DNA index system STRs. This report presents an updated validation of the PowerPlex 16 System on Applied Biosystem's 96 capillary 3730xl DNA Analyzer. The validation protocol developed in our laboratory allows for the analysis of 1536 loci (96 x 16) in c. 50 min. We have further optimized the assay by decreasing the reaction volume to one-quarter that recommended by the manufacturer thereby substantially reducing the total cost per sample without compromising reproducibility or specificity. This reduction in reaction volume has the ancillary benefit of dramatically increasing the sensitivity of the assay allowing for accurate analysis of lower quantities of DNA. Due to its substantially increased throughput capability, this extended validation of the PowerPlex 16 System should be useful in reducing the backlog of unanalyzed DNA samples currently facing public DNA forensic laboratories.     

TWGDAM validation of a nine-locus and a four-locus fluorescent STR multiplex system

The Gene Print PowerPlex 1.1/Amelogenin and FFFL Fluorescent STR Systems have been validated following the recommendations presented by the Technical Working Group on DNA Analysis Methods (TWGDAM). The PowerPlex 1.1/Amelogenin System supports simultaneous amplification of eight short tandem repeat loci and the Amelogenin gender identification marker. The loci D16S539, D7S820, D13S317, and D5S818 are labeled with fluorescein (FL) while the loci CSF1PO, TP0X, TH01, vWA and Amelogenin are labeled with carboxy-tetramethylrhodamine (TMR). The FFFL Multiplex System is composed of the loci F13A01, FESFPS, F13B, and LPL, each labeled with fluorescein. We have observed no overlap of alleles across loci labeled with an individual fluorescent dye. Samples of each system were amplified and labeled in a single reaction, separated by electrophoresis through a denaturing polyacrylamide gel, and amplified alleles detected using a Hitachi FMBIO Fluorescent Scanner. Alterations from the standard amplification protocols in cycle number and annealing temperature generally produced excellent results. In experiments testing sensitivity as little as 0.2 ng of DNA template could be detected. As expected, different body fluids from the same individuals generated identical DNA profile results. Template DNA derived from blood-strains deposited on a variety of matrix supports displayed robust amplification except for material derived from deposits on wood and Japanese orchid leaves. Mixtures of DNA templates could be interpreted with the minor component present in as little as ten percent of the total sample. Monoplex and multiplex amplifications produced identical amplified allele patterns, indicating that STR multiplex systems save template and increase efficiency in the amplification procedure without loss of quality. Analyses of genotype frequencies in African-American, Caucasian-American and Hispanic-American populations using all twelve loci were used to determine matching probabilities smaller than 1 in 1.14 x 10(8) and 1 in 2658 for the PowerPlex 1.1 and the FFFL Multiplex Systems, respectively. The matching probability achieved with the two systems combined is smaller than 1 in 3.03 x 10(11). The independence of alleles within loci was generally demonstrated by applying the exact test to demonstrate Hardy-Weinberg Equilibrium. All of the studies performed indicate that the PowerPlex 1.1/Amelogenin and FFFL Multiplex Systems are powerful, robust, and reliable investigative tools that can be used in the analysis of forensic samples.