DNA typing from skeletal remains using GlobalFiler™ PCR amplification and Investigator® 24plex QS kits

Since the beginning of our work in 2003 our laboratory has focused exclusively on STR DNA from bone, a powerful tool in missing person cases. In cases such as mass disasters or missing persons, human remains are challenging to identify as they may be fragmented, burnt, recovered from water, degraded, and/or contain inhibitory substances. To address these challenges, this study has evaluated the performance of relatively new STR kits Investigator® 24plex QS kit (Qiagen) and GlobalFiler™ PCR Amplification kit (Thermo Fisher Scientific) by comparing it with current uses of the AmpFLSTR® Identifiler® Plus kit (Applied Biosystems) to obtain genetic information from skeletal remains. We analyzed 20 bone samples of skeletal remains from routine casework submitted for body identifications by law enforcement corresponding using Investigator® 24plex QS kit and GlobalFiler™ PCR Amplification kit, previously analysed AmpFLSTR® Identifiler® Plus kit (Thermo Fisher Scientific). The data indicates that the STR profiles obtained using the GlobalFiler™ and Investigator® 24plex QS kit for analysis of skeletal remains has shown results in an increased number of reportable genetic loci, and provide greater power of discrimination in comparison to the Identifiler® Plus Kit. Advanced extraction and purification techniques, together with more sensitive and robust new amplification kits allowed us to overcome the challenges associated with processing compromised skeletal remains and ultimately obtain full STR DNA profiles in 99% of the bones.     

191名白山市汉族CODIS系统9个STR位点群体遗传学调查

目的　白山汉族CODIS系统 9个STR位点基因频率调查及其法医学应用 ;　方法　实验样本从 191位无相关白山市汉族个体获取 ,采用PCR复合扩增及 310遗传分析仪自动基因分型 ;　结果　这 9个STR位点均为高识别率位点 ,同重庆汉族人群群体遗传学数据比较显示有显著性差异 ;　结论　基因频率适合于白山汉族人群同一性概率及亲子鉴定概率计算 ;中国南北方汉族在个体识别及亲子鉴定概率计算时应采用本民族自己的等位基因频率。     

成都汉族、甘肃东乡族群体D10S1432和D10S1213 位点的遗传多态性分析

目的本研究的目的是了解人类基因组中D10S1432及D10S1213两个STR位点在成都汉族和甘肃东乡族群体中的遗传多态性分布及两个群体之间的关系。方法采用PCR、聚丙烯酰胺凝胶电泳及银染技术,共调查了209例样本。结果在D10S1432位点上观察到5个等位基因,15种基因型。在D10S1213位点上观察到9个等位基因,31种基因型。两位点的基因型频率在调查的两个群体中的分布符合Hardy-Weinberg平衡定律（P>0.05）。经统计,D10S1432在这两个群体中的杂合度为0.664和0.737,个人识别几率为0.827和0.820。D10S1213的杂合度为0.664和0.657,个人识别几率为0.836和0.882。结论结果表明,D10S1432和D10S1213两个位点在法医学个人识别和亲子鉴定中有较高应用价值。     

Successful STR and SNP typing of FTA Card samples with low amounts of DNA after DNA extraction using a Qiagen BioRobot EZ1 Workstation

FTA Cards (GE Healthcare) have been used for more than 4 years in Denmark for the collection of buccal cells as reference samples in crime cases. Semi-automated protocols for STR typing of DNA on punches of FTA Cards are routinely used. In average, full STR profiles were generated from approximately 95% of the FTA Cards with a standard punching protocol, while partial or no STR profile were obtained from 5% of the samples. Here, the Qiagen BioRobot^® EZ1 Workstation (Qiagen) and the EZ1 DNA Investigator Kit (Qiagen) was used to extract DNA from 29 FTA Cards from which a complete STR profile was not generated with the standard punching protocol. All 29 samples were successfully typed with the AmpF?STR^® Identifiler™ PCR Amplification Kit (Applied Biosystems) and with the SNPforID 49plex SNP assay. The lowest amount of DNA that resulted in complete STR and SNP profiles was 80 pg. The STR and SNP profiles were identical to those generated from another sample collected from each of the 29 individuals.     

24个Y-STR基因座等位基因频率的群体差异分析

目的调查华东地区汉族无关个体24个Y-STR基因座的群体遗传学多态性,比较华东汉族和广东汉族人群间的群体差异性。方法应用GFS 24Y STR荧光检测试剂盒,对华东地区268名汉族无关个体24个Y-STR基因座进行群体遗传学分析。比较华东汉族和广东汉族人群的等位基因频率,进行群体差异分析。结果华东地区268名汉族无关个体24个Y-STR基因座共检出235个等位基因,267种单倍型,GD值范围为0.564 9~0.966 8。华东汉族和广东汉族人群在DYS622、DYS552、DYS443等12个基因座的等位基因间的差异有统计学意义。结论 GFS 24Y STR荧光检测试剂盒中的Y-STR基因座具有良好的遗传多态性,可应用于父系亲缘关系鉴定。     

30个 InDels在中国三个民族的遗传学调查及法医学应用

目的调查Qiagen Investigator@ DIPplex试剂盒30个InDels多态性位点在中国汉族、藏族、维吾尔族人群中的群体遗传学数据,评估其法医学应用价值。方法采集汉、藏、维吾尔族各90名无关个体静脉血,提取DNA。使用3130xL毛细管电泳对该270份样品进行分型,通过统计计算相关的群体遗传学参数。结果实验得到270份样品的分型及基因型频率,30个InDels未明显偏离Hardy-Weinberg平衡及连锁平衡,在汉族、藏族、维吾尔族三个人群中的随机匹配概率分别为1.42×10（-11）、7.19×10（-12）、4.74×10（-13）,累积非父排除率（CPE）均大于0．9951。结论该组插入缺失位点在中国的汉族、藏族、维吾尔族人群中具有较高的多态性,能达到较高的个体识别能力,可以作为现有STR检验体系的补充。     

11个Y—STR基因座遗传多态性研究

目的 调查11个Y染色体STR基因座的遗传多态性。方法 利用PCR扩增和聚丙烯酰胺凝胶电泳技术对广州地区汉族群体无关男性血样进行11个Y—STR基因座的分型。结果 11个Y—STR基因座在广州地区汉族群体分别发现3～5个等位基因,GD值最低为0.3037(DYS434),最高为0.8455(DYS390)。结论 11个Y—STR基因座在广州地区汉族群体均具有较高的遗传多态性,可应用于法科学个体识别和亲子鉴定。     

Inferring ethnicity using 15 autosomal STR loci—Comparisons among populations of similar and distinctly different physical traits

The forensic utility of short tandem repeat (STR) polymorphisms for ethnic inference (EI) has recently received limited attention from the research community. The large volume of population specific STR data available from public literature affords us the opportunity to assess the reliability of EIs based on STR profiles, and present significant results. We applied a binary Bayesian approach using 15 autosomal STR loci from computer-simulated and real STR profiles of Han Chinese, Japanese, Korean, American Caucasian, and a South Asian Indian aboriginal tribal group called Lodha. A correct classification rate of ≥90% was achieved via simulation for each East Asian group (Han Chinese, Japanese, and Korean) when paired with the American Caucasian or the tribal Indian group. Similar result was obtained between the Caucasian and the Indian group, while the correct classification rates among the three East Asian groups were much lower (70%). Results based on real profiles closely matched those achieved via simulation, establishing the credibility of our classifier performance metrics. This study shows that binary Bayesian classifier performed best for ethnic groups with distinctly different physical traits. This observation might be due to accidental differences caused by bottlenecks, genetic drift, etc. However, it could also be taken as an indication that STRs, commonly referred to as “junk” DNA, may have an effect on phenotype, a possibility that warrants further research.     

Genetic polymorphisms of 20 STR loci in Chinese Han population in Tianjin,North China

The PowerPlex^® 21 System PCR Amplification Kit was a new PCR Amplification Kit developed for forensic laboratories, but there was a lack of data about this kit in Chinese people in Tianjin, North China. This kit contained 20 STR loci, D3S1358, D1S1656, D6S1043, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433 and FGA. In order to evaluate this kit and to get basic population data for its use in forensic practice in Chinese Han population, 360 unrelated Chinese Han individuals from Tianjin were typed using the Kit. Allele frequencies of the 20 STR loci and further population forensic genetic parameters were obtained. The observed genotype frequencies and expected genotype frequencies were evaluated by χ² test. No significant deviation from the Hardy–Weinberg equilibrium was observed in the population sample for the 20 STR loci. The population data in the present study can be used for routine forensic practice in Tianjin, North China.     

Comparisons between Japanese and Han Chinese populations for 261 autosomal STR loci

In this study, Japanese and Han Chinese individuals (n = 32, each) were genotyped for 261 autosomal STRs, and allele frequencies were calculated for each locus in each population. The average number of alleles for all loci in Japanese and Han Chinese populations was 6.65 and 6.56, respectively. The tests for deviations from HWE performed using an exact test showed that the number of STRs (P > 0.05) in Japanese and Han Chinese populations was 236 and 241, respectively. Calculation of forensic parameters showed heterozygosity, and the exclusion means in the Japanese population were 0.7185 and 4813 and those in the Han Chinese population were 0.7308 and 0.5008. In addition, population genetic analyses, such as principal component analysis and factorial correspondence analysis, were performed and a differential formula with likelihood ratios was applied for various number of STR loci based on the effectiveness of differentiation between the two populations. Accordingly, this study suggests that statistical differentiation between genetically close populations, such as the Japanese and Han Chinese populations, is possible if approximately 40–50 effective STR loci are analyzed.     

青岛地区汉族人群13个STR基因座的频率分布及法医学应用

目的　调查青岛地区汉族人群无关个体的 13个STR基因座 (D3S135 8、VWA、FGA、D8S1179、D2 1S11、D18S5 1、D5S818、D13S317、D7S82 0、D16S5 39、TH0 1、TPOX、CSFIPO)的基因频率分布 ,研究其遗传多态性及其在法医学个体识别及亲子鉴定中的应用价值。　方法　用美国ABI - 310型遗传分析仪对ProfilerPlus和Cofiler两个系统的 13个STR基因座的复合扩增产物进行毛细管电泳及四色荧光自动分析检测 ,基因分型软件为GeneScanv3.1和Genotyperv2 .5 .2。　 结果　获得 13个STR基因座在青岛地区汉族人群的基因频率分布数据 ,13个STR基因座的PIC >0 .5 ,DP >0 .71,CCE =0 .999999,TDP值接近 1,TPm =1.2× 10 -14 ,家系调查符合孟德尔遗传规律。　结论　ProfilerPlus和Cofiler两个系统的 13个STR基因座在法医学个体识别及亲子鉴定中具有较高的应用价值。     

汉族12个STR位点群体遗传学调查

目的240个汉族无关个体12个STR位点基因频率调查及其法医学应用;方法采用12位点复合扩增及变性聚丙烯酰胺凝胶电泳基因分型;结果该系统12个STR基因位点在汉族人群中均为高识别率位点,特别适合于陈旧血痕检验;结论12位点STR-PCR复合扩增系统检测方法简便,经济实用,在法医个体识别和亲子鉴定中具有应用价值。     

广东广西地区5个群体9个STR基因座的频率调查

目的 调查广东汉族、广西汉族、广西侗族、广西壮族、广西苗族5个群体9个STR基因座多态性,探讨其在法医学检验中的应用价值。方法 应用AmpFISTR Profiler PlusTM荧光标记复合扩增系统,对广东广西5个群体4个民族的1191个无关个体的血样DNA进行9个STR基因座的复合扩增;用ABI 3100遗传分析仪对扩增产物进行分型,统计9个STR基因座的群体遗传学参数。结果 9个STR基因座在广东广西地区5个群体中的累积偶合率为1.51×10-11～8.08×10-11,累积非父排除率为0.99981—0.99990。,结论 该9个STR基因座可满足汉族、壮族、侗族、苗族群体法医学的个体识别及亲权鉴定的需要。     

印记基因KCNQ1的遗传多态性及在亲权鉴定中的应用

目的为了调查印记基因KCNQ1的STR位点在中国汉族人群中的遗传多态性，利用亲源印记等位基因（parentally imprinting allele，PIA）分型法确定孩子的等位基因亲代来源，为亲权鉴定提供新的侯选STR位点。方法应用Chelex法提取153例佳木斯地区汉族健康无血缘关系个体DNA，用QIAamp Blood Kit（Qiagen）法提取3个家庭10个个体DNA，PCR扩增，凝胶电泳分型，ABIPRISM^TM 3730XL DNA测序仪测序；甲基化敏感性限制性内切酶消化孩子基因组DNA，PCR扩增，确定孩子等位基因的亲代来源。结果发现在中国佳木斯地区汉族人群中KCNQ1基因的STR有7个等位基因，多态信息含量为0．662，且KCNQ1基因的STR位点呈父源印记。结论印记基因KCNQ1的STR位点有很好的多态性，可为亲权鉴定提供新的侯选遗传标记，其亲源特异性甲基化标记有望应用于单亲鉴定中。     

西安汉族X染色体上6个STR位点的遗传多态性

目的调查西安汉族人群6个位于X染色体上的短串联重复序列:DXS8378、DXS7132、DXS6789、DXS101、HPRTB和DXS7423的基因及基因型频率分布。方法应用PCR和变性聚丙烯酰胺凝胶电泳及银染技术检测结果。结果在120例女性无关个体中,DXS8378、DXS7132、DXS6789、DXS101、HPRTB和DXS7423分别检出5、6、11、10、8和4个等位基因;分别检出10、17、29、32、22和7种基因型;此6个位点女性的基因型频率分布均符合Hardy-Weinberg平衡。结论此6个X染色体STR位点均有较高的个体识别率,在个体识别和女孩的亲权鉴定中有应用价值,对疾病相关研究有实际意义。     

19个STR在人群地域推断中的初步探索

本文汇总了近二十年19个常染色体STR(Short Tandem Repeat)在25个省份汉族人群中的研究报道。统计发现19个STR共有642个等位位点,其中等位位点数目最少的为D8S1179,有20个;数目最多的是D21S11,有60个等位位点。杂合度(He)为0.6203(TPOX)~0.9187(Penta E),多态性指数(PIC)为0.5600(TPOX)~0.9130(Penta E)个体识别率(PD)为0.6279(TPOX)~0.9859(Penta E)。19个STR的CPD、CPE和CMP分别为0.999999999999999999998、0.99999993和1.97×10^-21。通过POPTREE2.0对25个省份汉族19个STR进行聚类分析,发现我国汉族人群的分布有明显南北方地域差异,分为南方省份和北方省份。通过主成分分析也进一步证明了我国汉族人群分布具有南北方地域特点。另外,通过对15个少数民族与其所在省份汉族人群和无关省份汉族人群的STR聚类分析,发现新疆维吾尔族、哈萨克族与新疆汉族聚类在一个亚分支中;广西汉族、云南汉族与该省份的少数民族聚类在一起,这也进一步证明了我国汉族人群和少数民族具有一定的地域分布特性。综上所述,STR不仅可以应用于个体识别、亲子鉴定等,未来还可用于人员地域推断。伴随着STR数量的不断增加和人员STR数据库的不断丰富以及与测序技术的结合,STR技术将会在各类案件中发挥更多作用。     

荧光标记复合扩增检测4个STR基因座多态性

目的建立荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座检测分型方法,并对成都汉族群体4个基因座的遗传多态性进行调查。方法用6-FAM标记D1S2142和D15S659引物,HEX、TMR分别标记D14S306和D13S1492引物,PCR复合扩增,310基因分析仪电泳自动收集电泳结果数据,GeneScan Analysis Software3.7NT软件计算扩增产物片段相对大小,Genotyper(3.7NT软件进行样本基因型分型,建立了荧光标记复合扩增检测4个STR基因座基因型的方法,对145名成都汉族无关个体样本进行分型。结果荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座,每个STR基因座都获得了清晰的基因型分型结果。145份样本,4个STR基因座分别检出10,14,7,12个等位基因和22,54,21,39种基因型,其基因型分布均符合Hardy-W e inberg平衡。4个基因座在成都汉族群体的杂合度分别依次为0.7793,0.8345,0.7793和0.8345;多态信息含量分别依次为:0.7656,0.8730,0.7470和0.8312。累计非父排除率为0.9783,累计个人识别机率为0.9999 917。结论荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座,可实现对每个基因座准确分型;成都汉族群体该4个基因座的遗传学数据,可为群体遗传学和法医学研究与应用提供基础资料。     

2个Y-STR基因座在成都汉族群体中的遗传学研究

目的　为了寻求新的适合于法医学应用的Y染色体STR基因座 ,我们调查了基因座DYS44 2和DYS44 6在成都群体中的分布。　方法　样本来自于成都地区汉族无血缘关系的个体 ,通过Chelex法提取样本DNA ,利用PCR扩增硝酸银染色方法进行分型。　结果　DYS44 2是一个四核苷酸简单重复基因座 ,而DYS44 6则为五核苷酸简单重复基因座。男性样本都出现了谱带 ,而女性样本则无PCR产物。DYS44 2基因座和DYS44 6基因座变异度分别为 :0 .6 86 7、0 .75 5 2。　结论　DYS44 2和DYS44 6是非常适合于法医学应用的STR基因座。     

中国武汉汉族群体STR D13S325基因座的多态性研究

应用PCR ,聚丙稀酰胺凝胶垂直电泳及银染技术对 2 16例中国汉族人群STRD13S32 5基因座进行了多态性研究 ,首次获得了中国武汉汉族人群基因频率分布资料。检出 11个等位基因 ,30个基因型 ,基因频率分布符合Hardy Weinberg平衡。DP值为 0 92 92 ,三联体PE值 0 6 137,二联体PE值为 0 4370 ,PIC值为 0 775 5。观察 2 0 0次减数分裂未发现突变基因。研究证实D13S32 5是一个高多态性STR基因座标记系统。     

6个五核苷酸STR基因座荧光复合扩增体系的建立

目的 构建6个五核苷酸STR基因座荧光复合扩增体系。方法筛选6个多态性程度较高的五核苷酸STR基因座D10S2325、Penta B、Penta W、PentaX、Penta D和PentaE,按照复合扩增引物设计要求,重新设计引物并标记荧光染料,经反复调整和优化,构建6基因座荧光复合扩增体系,并用该复合扩增体系对239名武汉汉族无关个体进行分型。结果6个五核苷酸STR基因座荧光复合扩增体系分型稳定,可重复性好,与各自相应单基因座分型结果完全一致;累积个人识别率达0．999999988,累积非父排除率达0．998063807。结论本文构建的6个五核苷酸STR基因座荧光复合扩增体系具有很高的法医学实用价值,可作为商品化试剂盒的有效补充。