A 26plex Autosomal STR Assay to Aid Human Identity Testing*†

Abstract: A short tandem repeat multiplex assay has been successfully developed with 25 autosomal loci plus the sex‐typing locus amelogenin for a total of 26 amplified products in a single reaction. Primers for the loci were designed so that all of the amplicons present were distributed from 65 base pairs (bp) to less than 400 bp within a five‐dye chemistry design with the fifth dye reserved for the sizing standard. A multiplex design strategy was developed to overcome challenges encountered in creating this assay. The limits of the multiplex were tested, resulting in the successful amplification of a wide range of genomic DNA sample concentrations from 2 ng to as low as 100 pg with 30 cycles of PCR. The 26plex has the potential to benefit the forensic community for reference sample testing and complex relationship evaluation.     

Characterization of 26 miniSTR loci for improved analysis of degraded DNA samples

An additional 20 novel mini-short tandem repeat (miniSTR) loci have been developed and characterized beyond the six previously developed by our laboratory for a total of 26 non-CODIS miniSTR markers. These new markers produce short PCR products in the target range of 50-150 base pairs (bp) by moving the primer sequences as close as possible-often directly next to the identified repeat region. These candidate loci were initially screened based on their small amplicon sizes and locations on chromosomes currently unoccupied by the 13 CODIS STR loci or at least 50 Mb away from them on the same chromosome. They were sequenced and evaluated across more than 600 samples, and their population statistics were determined. The heterozygosities of the new loci were compared with those of the 13 CODIS loci and all were found to be comparable. Only five of the new loci had lower values than the CODIS loci; however, all of these were much smaller in size. This data suggests that these 26 miniSTR loci will serve as useful complements to the CODIS loci to aid in the forensic analysis of degraded DNA, as well as missing persons work and parentage testing with limited next-of-kin reference samples.     

河南汉族群体D6S1043和D12S391遗传多态性

本文调查了D12S391和D6S1043基因座等位基因频率在231名河南汉族群体中的分布,以期为这两个基因座在相关汉族群体内的应用提供基础数据。     

新疆维吾尔族群体D6S1043、D12S391基因座遗传多态性

本研究调查了居住在新疆境内的677个维吾尔族无关个体D6S1043、D12S391基因座的遗传多态性,现报道如下。 1材料与方法 677份无关个体血样全部来自于日常检案,采耳垂静脉血制成血痕样本保存。血样采取Chelex-100法提取。     

苏南地区汉族人群D2S1772和D15S659基因座遗传多态性

<正>D2S1772和D15S659短串联重复序列分别定位于人类第2号、第15号染色体,核心序列分别为 (GATA)n、(GATA)n,由Marshfield实验室从人类基因组分离获得(http:／／www．chic．org)。本文应用聚丙烯酰胺垂直电泳及银染方法,获得了中国苏南地区汉族人群这两个基因座的等位基因频率。     

福建汉、畲族D6S1043和D12S391基因座遗传多态性

D12S391和D6S1043基因座分别位于人类第12号和第6号染色体上，核心序列分别为（ATCT）。和（AGAT）x（AGAC）Y（AGAT）z。本文采用Sinofiler试剂盒对福建汉族和畲族上述2个基因座遗传多态性进行调查，为相关检测提供基础数据。     

武汉汉族人群短串联重复序列D5S818,D7S820位点多态性研究

目的 研究Ｄ５Ｓ８１８,Ｄ７Ｓ８２０的多态性及法医学应用价值。方法 应用聚合酶链反应,聚丙烯酰胺凝胶电泳分离及银染是带技术对地区汉族２３２例无关人体作Ｄ５Ｓ８１８,Ｄ７Ｓ８２０位点分型调查。结果 Ｄ５Ｓ８１８和Ｄ７Ｓ８２０位点分别检了８个和６个等位基因,获汉族人群基因频率分布。二位点基因型频率分布符合Ｈａｒｄｙ Ｗｅｉｎｂｅｒｇ平衡。     

3个miniSTR基因座荧光标记复合扩增体系的建立及应用

目的建立扩增片段小于115 bp,包括D1S1676、D6S1274和D17S1299 3个非CODIS系统的miniSTR基因座复合扩增系统,用于高度降解DNA样本的基因分型。方法采用不同荧光染料标记引物,通过PCR扩增,利用310遗传分析仪对100份成都汉族健康无关个体血样以及2份高度降解检材进行检测。结果荧光标记复合扩增D1S1676、D6S1274和D17S1299 3个miniSTR基因座,每个基因座均获得了清晰的基因型分型结果。100份样本,3个miniSTR基因座分别检出个9、9、7个等位基因和27、23、18种基因型,基因型分布均符合Hardy-Weinberg平衡。3个基因座在成都汉族人群的累积非父排除率、累积个体识别能力分别为0.9991和0.9160。结论本系统可以应用于个体识别和亲权鉴定,为DNA高度降解样本分型提供了新的方法。     

荧光标记3个miniSTR基因座复合扩增系统的建立

目的建立扩增片段<135bp,包括D5S818,D8S1179,D16S539 3个miniSTR基因座复合扩增系统。方法采用不同荧光染料标记引物,通过PCR扩增,利用ABI 3100遗传分析仪进行片段长度分析,对100份无关个体血样,10个家系样本以及30份高度降解检材进行检测。结果本系统DNA分型结果与AmpFLSTR Identifiler试剂盒完全一致,且灵敏度高于AmpFLSTR Identifiler试剂盒。结论本系统可以应用于个人识别和亲权鉴定,为降解DNA样本分型提供了新的方法。     

Developmental validation of the AmpFℓSTR® Identifiler® Plus PCR Amplification Kit: an established multiplex assay with improved performance

Abstract: Analysis of length polymorphism at short tandem repeat (STR) loci utilizing multiplex polymerase chain reaction (PCR) remains the primary method for genotyping forensic samples. The AmpF?STR^® Identifiler^® Plus PCR Amplification Kit is an improved version of the AmpF?STR^® Identifiler^® PCR Amplification Kit and amplifies the core CODIS loci: D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, TH01, TPOX, and vWA. Additional loci amplified in the multiplex reaction are the sex‐determinant, amelogenin, and two internationally accepted loci, D2S1338 and D19S433. While the primer sequences and dye configurations were unchanged, the AmpF?STR^® Identifiler^® Plus PCR Amplification Kit features an enhanced buffer formulation and an optimized PCR cycling protocol that increases sensitivity, provides better tolerance to PCR inhibitors, and improves performance on mixture samples. The AmpF?STR^® Identifiler^® Plus PCR Amplification Kit has been validated according to the FBI/National Standards and Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The validation results support the use of the AmpF?STR^® Identifiler^® Plus PCR Amplification Kit for human identity and parentage testing.     

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…     

河北汉族群体D17S974和D11S4463miniSTR遗传多态性

STR技术目前已经广泛应用于法医学实践,但在实际检察中,一些微量及降解的检材,在使用商品化STR复合扩增试剂盒进行检测时,片段较大的STR基因座常因出现“优势扩增”或“无效扩增”而不能成功得出结论。miniSTR分析技术是尽可能靠近核心序列设计引物以缩短扩增片段长度（〈125bp）,可提高高度降解DNA样本的检测成功率。     

武汉汉族人群短串联重复序列D5S818， D7S820位点多态性研究

目的研究Ｄ５Ｓ８１８,Ｄ７Ｓ８２０的多态性及法医学应用价值。方法应用聚合酶链反应（ＰＣＲ）、聚丙烯酰胺凝胶电泳分离及银染显带技术对武汉地区汉族２３２例无关个体作Ｄ５Ｓ８１８,Ｄ７Ｓ８２０位点分型调查。结果Ｄ５Ｓ８１８和Ｄ７Ｓ８２０位点分别检出８个和６个等位基因,获汉族人群基因频率分布。二位点基因型频率分布符合ＨａｒｄｙＷｅｉｎｂｅｒｇ平衡。位点杂合度分别为０８１２１和０７９３４,个人识别能力分别为０９４１６和０９２５５,非父排除率分别为０５８４２和０５８１６。结论Ｄ５Ｓ８１８和Ｄ７Ｓ８２０ＳＴＲ位点均是高杂合度、高鉴别能力的遗传标记系统,在法医学个人识别和亲子鉴定中有较高实用价值     

Development and validation of the AmpFℓSTR® Identifiler® Direct PCR Amplification Kit: a multiplex assay for the direct amplification of single-source samples

Abstract: The AmpF?STR^® Identifiler^® Direct PCR Amplification Kit is a new short tandem repeat multiplex assay optimized to allow the direct amplification of single‐source blood and buccal samples on FTA^® card without the need for sample purification and quantification. This multiplex assay has been validated according to the FBI/National Standards and SWGDAM guidelines. Validation results revealed that slight variations in primer concentration, master mix component concentration, and thermal cycling parameters did not affect the performance of the chemistry. The assay’s sensitivity was demonstrated by amplifying known amounts of white blood cells spotted onto FTA^® cards, and the assay’s specificity was verified by establishing minimal cross‐reactivity with nonhuman DNA. No effect on the age of the sample stored on the FTA^® substrate was observed and full concordance was established in the population study. These findings of the validation study support the use of the Identifiler^® Direct Kit for forensic standards and database samples genotyping.