利用峰面积分析混合DNA样品

在法医DNA检验过程中 ,经常会遇到多个体的混合样本 ,如受害人和嫌疑人的混合样品 ,几个嫌疑人的混合样品。荧光标记自动分析STR系统 ,不仅能根据位点等位基因的个数判断样品是否为混合样品 ,而且还能根据等位基因峰面积 (谱带荧光总强度 )确定混合样品中各组分的混合比例 ,推断混合样品中各组分的基因分型[1] 。Gill等建立了利用峰面积分析混合样品的方法[2 ] 。本文利用该法对一宗案件中的混合样品进行了分析 ,推断混合样品各组分的比例及各组分的基因分型。材料和方法2 0 0 1年于中山某地发生一宗强奸案 ,送检材料为事主血纱 ,嫌疑人 …     

利用峰面积分析混合DNA样品

在法医DNA检验过程中，经常会遇到多个体的混合样本，如受害人和嫌疑人的混合样品，几个嫌疑人的混合样品。荧光标记自动分析STR系统，不仅能根据位点等位基因的个数判断样品是否为混合样品，而且还能根据等位基因峰面积(谱带荧光总强度)确定混合样品中各组分的混合比例，推断混合样品中各组分的基因分型。Gill等建立了利用峰面积分析混合样品的方法。本文利用该法对一宗案件中的混合样品进行了分析，推断混合样品各组分的比例及各组分的基因分型。     

SNP-STR遗传标记复合扩增体系的构建及法医学应用

目的筛选并构建与目前STR数据库兼容的SNP-STR遗传标记复合扩增体系,调查其在四川汉族群体中的遗传多态性,并探讨其在混合DNA样本分析中的应用价值。方法以现有商业试剂盒中使用的STR遗传标记为基础,筛选与STR遗传标记相邻的SNP位点并组成SNP-STR遗传标记。运用SNP等位基因设计特异性引物,构建基于等位基因特异性扩增的SNP-STR遗传标记复合扩增体系。调查该体系在四川汉族群体的遗传多态性,并评价不同位点数目的体系对两个体混合DNA样本的检测效能。结果筛选并构建了由13个SNP-STR遗传标记构成的等位基因特异性复合扩增体系。在四川汉族群体中,各位点杂合度为0.76～0.88,累积个体识别率达0.999 999 999 999 999 968。在对两个体混合DNA的分析中:单位点扩增时,混合样本的混合比例达到1 000∶1时依然可以检测到少量成分所特有的分型;多位点复合扩增时,混合比例最大可达500∶1;随着体系中位点数量的增加,对混合DNA中少量成分的检测效能降低。结论SNP-STR遗传标记较STR具有更高的多态性,其构成的复合扩增体系对混合样本的分析效能优于传统的STR复合扩增体系。     

四类遗传标记的检测用于混合精斑的个人识别

目的探讨综合应用常染色体STR与Y-STR、X-STR、mtDNA高变区Ⅰ序列检测四类遗传标记,提高两男性混合精液样品的个人识别率。方法使用商品化试剂盒检测混合精液样品的常染色体与X、Y染色体STR,应用单链构象多态性(single strand conformation polymorphism,SSCP)法分离两男性混合精液样品的线粒体DNA(mitochondrial DNA,mtDNA)高变区Ⅰ序列,并进行序列分析。结果混合精液样品比例超过1:10(高组分所占比例继续增加而低组分所占比例继续减少)时可以确定高拷贝者的全部四类遗传标记系统的分型。比例接近时,通过与嫌疑人检测结果的比较可获得排除或基本认定的意见。结论四类遗传标记系统的联合检测有助于提高两男性混合精液样品的个人识别率。     

两个体混合样本比例的PCR-STR检测研究

目的探讨PCR-STR检测两个体混合样本比例的灵敏度。方法按不同比例将两个体全血、单个核细胞及纯DNA 3种不同的样本进行混合,应用AB I公司生产的profile p lus商品化试剂盒多重PCR扩增后,用AB I3100基因分析仪进行毛细管电泳,检测基因座及其峰面积,并就混合样本中两单个核细胞样本DNA含量百分比与扩增前混合样本比例的关系进行相关分析。结果当样本混合比例为4%+96%时,可明确区分混合样本中两个体的基因型,且扩增前混合细胞百分率与扩增后两者峰面积比值呈直线相关。结论PCR-STR检测两个体混合样本比例的灵敏度有一定范围,并可进行半定量。     

基于二代测序技术的多源遗传标记检测体系的构建与验证

近年来，法医实践中对复杂亲缘关系的鉴定需求越来越多，需要联合多种遗传标记进行判断，如STR、X/Y遗传标记、SNP、线粒体DNA等。二代测序技术可以将多种遗传标记纳入一个检测体系中。本文报道了开发出的一套包含29个常染色体STR、36个Y-STR、32个X-STR、71个Y-SNP以及mtDNA全基因组的检测体系DNATyper^TMNGSPanel v1.0。根据DNA分析方法科学工作组(SWGDAM)的验证指南，对该体系的重复性、准确性、一致性、灵敏度、混合样本、物种特异性等指标进行了评估。结果表明，该体系分型结果与毛细管电泳的一致性为99.72%，与ForenSeq?DNA Signature Prep Kit试剂盒相重合的基因座结果均完全一致。在0.5～10 ng DNA模板量范围内无等位基因丢失，而在0.25、0.125 ng时分别出现2、9个基因座的丢失。在男女混合比例为2︰1时女性成分开始出现等位基因丢失，混合比例为9︰1、4︰1、2︰1、1︰1时，检出率分别为54.72%、81.13%、98.11%、100%，若男女混合比例为1︰4则男性成分开始出现...     

混合生物样品的组分分析及其STR基因型判定

目的应用荧光标记STR多基因座联合检测技术对混合生物样品较少组分最低比例检出限和STR基因型的判定进行研究。方法 将已知浓度的人标准细胞系DNA:9947A和K562分别按照1:1、1:4、1:9、1:19、1:39、1:59、1:79和1:99比例混合后作为扩增模板,应用Profiler plus试剂盒对D3S1358、VWA、FGA、D8S1179、D21S11、D18S51、D5S818、D13S317、D7S820和性别鉴定基因座检验。结果在比例为1:19时能明确判定两个体各基因座基因型,且两样品基因型结果峰高平均值之比与样品浓度之比正相关。结论应用荧光标记STR多基因座联合检测技术可以对一定比例的混合生物样品进行STR基因型判定,并对其混合比例状况进行大致推断。     

单细胞分离荧光原位杂交法用于男女混合血DNA分型

目的采用单细胞分离荧光原位杂交法精确分离混合血样中男性和女性细胞并进行分型检验。方法收集男、女性血,按照男∶女为1∶5、1∶10、1∶20制备混合血样,加入0.075mol/L KCl 600μL,轻混、放置30min后加入150μL固定液离心留沉淀涂片,利用Vysis 30-161050试剂盒进行荧光原位杂交,并用PALM激光显微捕获系统分离出男、女性细胞,使用Identifiler试剂盒复合扩增并进行检测。结果捕获8个血细胞即可得到完整的DNA分型,且随着细胞数目的增多,检出率逐渐提高而等位基因丢失率逐渐降低。10个血细胞的检出率最高,为93.75%。5μL男性血液与本实验各比例女性血混合用本文方法检验均可获得男性分型。案例混合血斑经检验获得单一男性和女性分型。结论单细胞分离荧光原位杂交法可用于男女混合血样本中DNA分型检验。     

法医学二代测序STR分型准确度与测序深度的关联性评估

目的利用实验数据对法医学二代测序STR分型测序深度与分型结果准确度的关联性进行评估。方法使用商业化基因组DNA制备单一来源和混合的DNA样本,以Thermo Fisher公司的25重早期测试试剂盒进行目的STR片段扩增,每种扩增产物分别使用4种不同的序列标签平行建库,并控制标记每一种序列标签的文库上机量依次占一张Ion 318芯片的1/4、1/8、1/16、1/32。经Ion PGMTM基因测序仪测序,以及Ion Torrent SuiteTM软件进行数据分析;同时对庞敬博等人发表的基于相同试剂盒和测序仪检测的95名中国汉族无关个体的6928条等位基因、影子峰和噪音序列进行测序深度统计分析,寻找测序深度与STR分型准确度的关联性。结果各基因座测序深度随文库上样量减少而呈明显下降趋势。对于单一来源样本,每张芯片上样不超过8个均一化文库可实现全部基因座的完整分型;对于1∶20比例的混合DNA,每张芯片上样不超过4个均一化文库时,未发现微量组分的等位基因丢失。人群数据测序深度统计显示,该体系基因座间存在不均衡性,有必要针对各基因座分别设定分析阈值参数。结论测序深度与法医学STR分型结果的准确性密切相关,各基因座最低测序深度与平均测序深度的比值可作为设定分析阈值的重要参考指标。本研究确定的单张芯片上样数量仅适用于本实验体系,但相关实验设计和方案可供其他实验体系开展类似工作参考。     

STRtyper-10G系统9个STR基因座突变分析

目的观察和分析STRtyper-10G系统9个STR基因座的突变特点。方法在7 707例肯定亲子关系的案件中,统计使用STRtyper-10G试剂盒（9个STR基因座）检测发现的突变事件,判断突变等位基因的来源,计算各基因座的突变率,分析突变特点。结果在9个基因座上共发现118个突变事件,均为1步突变;平均突变率为1.69×10-3（95%CI 1.40×10-3~2.03×10-3）,各基因座的突变率介于0.78×10-3~2.84×10-3,父、母来源突变比例为9.64∶1;短、中、长等位基因的突变比值约为1∶8∶3,增加和减少重复单位的突变比值为1.29∶1。结论 9个基因座的突变率存在显著差异,实际检案时应结合各基因座的突变率进行PI值计算更为科学。     

荧光标记STRs复合扩增分析混合血样品

探讨应用荧光标记STRs复合扩增技术能够检测出混合血样品中较少个体成份的最低检出量及所占的比例多少与基因型的峰值高低是否存在一定的剂量反应关系。采用荧光标记STRs复合扩增技术 ,分析 4对两无关男 /女的混合血样品。扩增基因座包括D8S1179、D2 1S11、D18S5 1、D3S135 8、vWA、FGA、D5S818、D13S317、D7S82 0及性别Amelogeine。结果表明 ,对经用酚 /氯仿有机溶剂方法提取的混合血样品中较少成份的最低检出量为 ,能够从10ng混合DNA中检出 1ng的较少成份 ;从 10∶90至 5 0∶5 0 5个不同比例组 ,较少成份所占比例多少与其对应基因型峰值的高低呈现一定的正相关趋势。应用荧光标记STRs复合扩增技术可能较好地解决混合血样品的个体识别问题     

Estimating Individual Contributions to Complex DNA SNP Mixtures.,

High‐throughput sequencing (HTS) of large panels of single nucleotide polymorphisms (SNPs) provides an alternative or complimentary approach to short tandem repeats (STRs) panels for the analysis of complex DNA mixture forensic samples. For STRs, methods to estimate individual contribution concentrations compare capillary electrophoresis peak heights, peak areas, or HTS allele read counts within a mixture. This article introduces three approaches (mean, median, and slope methods) for estimating individual DNA contributions to forensic mixtures for HTS/massively parallel sequencing (MPS) SNP panels. For SNPs, the major:minor allele ratios or counts, unique to each contributor, were compared to estimate contributor proportion within the mixture using the mean, median, and slope intercept for these alleles. The estimates for these three methods were typically within 5% of planned experimental contributions for defined mixtures.     

Variant alleles on the penta E locus in the PowerPlex 16 kit

Penta E in the PowerPlex 16 kit is a pentanucleotide tandem repeat marker located on Chromosome 15, containing an AAAGA repeat motif. Variant alleles (18.4 and 19.4) were found in the Japanese population. A sequence analysis revealed that both the variant alleles had a partial repeat motif of AAAA, resulting in one-base-shorter alleles compared to known alleles. Despite the relatively large amplicon sizes (379 to 474 bp) of Penta E, an accurate allele assignment can be reliably made by capillary electrophoresis. However, alleles differing in size by only one base (e.g., 18.4 and 19) were not separated and appeared as a single broad peak. The Genotyper software assigned one of the component alleles to this peak. Therefore, such broad peaks require careful interpretation so as to not overlook the other component allele contained by the peak. As an index to recognize a peak containing two alleles, the ratio of peak area to peak height was found to be useful.     

STR和性别位点PCR产物的荧光自动检测

运用荧光染料标记引物、变性聚丙烯酰胺凝胶电泳 (denaturingpolyacrylamidegelelec trophoresis,denaturingPAGE)结合荧光法检测6个STR位点 (vWA31A、TH01、F13A1、FES、TPOX、CSF1PO)及1个性别鉴定位点(Amelogenin) ,就荧光自动检测的重复性、准确性、分辩力等方面进行了研究 ,建立了6个STR位点等位基因判定视窗及用荧光DNA测序仪进行STR分析的方法 ,实现电脑自动判读结果。结果表明 ,本文所述STR和性别鉴定的自动检测方法可靠、分辨力高 (可达1bp) ,所得数据有助于荧光自动DNA分型软件系统的数据积累。     

Multiplexing of Y chromosome specific STRs and performance for mixed samples

A combination of four Y-specific polymorphic STR loci was amplified simultaneously using fluorescently labeled primers. Multiplex conditions required optimization to eliminate constant bands and amplification products for female DNA. A series of experiments was carried out for mixtures of DNA from two males, and from male and female individuals for the Y-specific STRs and an autosomal locus. For the male/male mixtures amplified with the Y specific system, and amplified for an autosomal locus, the minor component in the mixture could only be identified up to a ratio of 1:10, 1:50 respectively. In male/female DNA mixtures the Y STR alleles could be identified for the highest ratio tested, 400 pg male in DNA in 800 ng female DNA which amounts to a ratio of 1:2000.     

Natural DNA mixtures generated in fraternal twins in utero

Analysis of multiple genetic loci using short tandem repeats (STR) is widely used in human identity testing because the extensive polymorphism at these loci allows for a high degree of discrimination among individuals. We recently received a forensic case that included several pieces of evidence and reference blood samples. Upon initial testing, one of the suspects had a DNA profile that included three alleles at four of the nine loci tested (vWA, FGA, TH01, and D5S818). At each locus, two of the alleles appeared to be "major" alleles with a third "minor" allele present. The profile appeared to be a mixture of two people. Contamination of this first reference sample was suspected and a second, unopened blood specimen was requested from this individual. The DNA profile from this second reference specimen was identical to that of the original specimen at each locus. One of the evidence samples also displayed an identical mixed DNA profile matching that of the reference specimens mentioned above. The relative peak heights of the two "major" and one "minor" allele remained constant in all three samples. Additional background information revealed that the suspect had not received a bone marrow transplant or blood transfusion. However, it was disclosed that this individual is a fraternal (dizygotic) twin. We hypothesize that an exchange of blood cells between the fetuses occurred in utero and that the additional alleles present in these reference samples are derived from cells contributed by his twin sibling. No additional specimens from the suspect or his twin could be obtained for confirmation, and our hypothesis remains untested. Forensic scientists should be aware of this possibility when faced with a DNA profile in which extra alleles at multiple loci are detected.     

Internal validation of the AmpFlSTR Yfiler amplification kit for use in forensic casework.

Y-chromosomal short-tandem repeat (Y-STR) amplification has been used in forensic casework at the Bureau of Criminal Apprehension (BCA) Forensic Science Laboratory since 2003. At that time, two separate amplifications were required to type the SWGDAM recommended loci (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438, and DYS439). The Yfiler kit coamplifies these loci as well as DYS437, DYS448, DYS456, DYS458, DYS635, and Y GATA H4. The Yfiler kit was validated following the internal validations outlined in the SWGDAM revised validation guidelines. Our studies show that 0.125 ng of male DNA will generate a complete 17 locus profile and that as little as 0.06 ng of male DNA yields an average of nine loci. In the male-male mixtures, a complete profile from the minor component was detected up to 1:5 ratio; most of the alleles of the minor component were detected at a 1:10 ratio and more than half the alleles of the minor component were detected at a 1:20 ratio. Complete YSTR profiles were obtained when 500 pg male DNA was mixed with female DNA at ratios up to 1:1000. At ratios of 1:5000 and 1:10,000 (male DNA to female DNA) inhibition of the YSTR amplification was evident. The YSTR results obtained for the adjudicated case samples gave significantly more information than the autosomal results. Our studies demonstrate that the Yfiler kit is extremely sensitive, does not exhibit cross-reactivity with female DNA, successfully types male DNA in the presence of overwhelming amounts of female DNA and is successful in typing actual forensic samples from adjudicated cases.     

Internal Validation of the AmpFlSTR Yfiler™ Amplification Kit for Use in Forensic Casework

Abstract:  Y-chromosomal short-tandem repeat (Y-STR) amplification has been used in forensic casework at the Bureau of Criminal Apprehension (BCA) Forensic Science Laboratory since 2003. At that time, two separate amplifications were required to type the SWGDAM recommended loci (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS438, and DYS439). The Yfiler™ kit coamplifies these loci as well as DYS437, DYS448, DYS456, DYS458, DYS635, and Y GATA H4. The Yfiler™ kit was validated following the internal validations outlined in the SWGDAM revised validation guidelines. Our studies show that 0.125 ng of male DNA will generate a complete 17 locus profile and that as little as 0.06 ng of male DNA yields an average of nine loci. In the male–male mixtures, a complete profile from the minor component was detected up to 1:5 ratio; most of the alleles of the minor component were detected at a 1:10 ratio and more than half the alleles of the minor component were detected at a 1:20 ratio. Complete YSTR profiles were obtained when 500 pg male DNA was mixed with female DNA at ratios up to 1:1000. At ratios of 1:5000 and 1:10,000 (male DNA to female DNA) inhibition of the YSTR amplification was evident. The YSTR results obtained for the adjudicated case samples gave significantly more probative information than the autosomal results. Our studies demonstrate that the Yfiler™ kit is extremely sensitive, does not exhibit cross-reactivity with female DNA, successfully types male DNA in the presence of overwhelming amounts of female DNA and is successful in typing actual forensic samples from adjudicated cases.