中国汉族人群 8个 STR位点荧光标记同步检测及其频率分布

目的 对血液等微量生物学检材进行 8个 STR多态性位点及一个性别鉴定位点的复合检测 ,并调查了 350名中国汉族无关个体上述基因位点等位基因分布情况。方法 所选位点为 vWA、 TH01、 TPOX、 CSF1PO、 D5S818、 D13S317、 D7S820、 D16S539及性别鉴定位点 Amelogenin,应用荧光染料标记引物,利用 PE－ 377 DNA片段分析仪对扩增产物进行基因分型。结果 共检出 63个等位基因及两个性别决定基因 ,总鉴别机率（ TDP）值达 99.999 999 98％,对法医学常见极微量生物学检材的检测获得成功, DNA模板需要量为 0.5～ 1.0ng,通过家系调查,证明上述位点遗传稳定,符合孟德尔遗传规律。结论 上述 8个 STR多态性位点具备了个体认定能力 ,是对微量生物学检材进行个体识别鉴定的理想方法。     

犬17A STR 5色荧光检测试剂盒在警犬DNA鉴定中的应用研究

目的建立犬DNA检测方法。方法采用自主研发的多重PCR扩增体系和五色荧光（FAM、HEX、TAMRA、ROX和SIZ）自动化检测技术调查了犬DAmel和VWFX等16个STR基因座和1个性别决定基因座多态性,并计算该16个STR基因座的等位基因频率（P）、杂合度（H）、多态信息含量（PIC）和非父排除率（PE）。实验犬为11个品种231头犬。结果结果显示该16个STR位点的非父排除率（PE）和个体识别力（DP）分别为0.995026和0.999999992。结论显示研发的犬STR-DNA荧光检测试剂盒可作为犬DNA鉴定常规应用。     

二代测序试剂盒SNP位点遗传学参数和对比

目的计算二代测序试剂盒SNP位点的遗传学参数,与STR基因座进行对比,以建立SNP和STR的系统效能换算比例。方法对二代测序试剂盒(Foren Seq~(TM)DNA Signature Prep试剂盒和Precision ID Identity Panel试剂盒)共101个SNP位点进行Hardy-Weinberg平衡检验,计算SNP位点在个体识别、标准三联体鉴定、二联体鉴定及双亲皆疑鉴定中的各项系统效能参数,并与STR基因座进行对比。结果除无基因型频率数据的2个位点外,99个SNP位点符合Hardy-Weinberg平衡检验(P0.05)。Foren Seq~(TM)DNA Signature Prep试剂盒94个SNP位点的累积个体识别率(cumulative discrimination power,CDP)为1-1.152 1×10~(-34),累积三联体非父排除率(cumulative probability of exclusion in trios,CPE_(trio))为1-4.416 9×10~(-8),累积二联体非父排除率(cumulative probability of exclusion in duos,CPE_(duo))为1-8.483 7×10~(-5),双亲皆疑累积排除率(cumulative probability of exclusion in alleged parents cases,CPE_(AP))为1-1.222 7×10~(-12)。Precision ID Identity Panel试剂盒90个SNP位点的CDP为1-2.052 4×10~(-33),CPE_(trio)为1-8.709 3×10~(-8),CPE_(duo)为1-1.1638×10~(-4),CPE_(AP)为1-3.725 7×10~(-12)。在个体识别、标准三联体鉴定、二联体鉴定、双亲皆疑鉴定中,分别平均有2.85、4.51、4.88、4.55个SNP位点等于1个STR基因座的系统效能。结论二代测序试剂盒SNP位点的系统效能较高,多个SNP位点联合可应用于法庭科学中的个体识别和亲子鉴定。     

河南汉族人群39个STR基因座遗传多态性

本研究采用AGCU Ex22和21＋1荧光标记复合扩增系统对河南汉族人群39个STR 基因座的遗传多态性进行调查,获得了这39个STR基因座的河南汉族人群遗传学数据,旨在为法医学个人识别、亲权鉴定、法医DNA数据库提供基础数据。     

印记基因KCNQl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KCNQl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 l(jt(Qiagen)法提取3个家庭10个个体DNA,PCR扩增,凝胶电泳分型,ABlPRIsMTM3730xL 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KCNQI基因的STR位点呈父源印记.结论 印记基因KCNQl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.     

联合应用多个DNA位点进行尸源鉴定

目的　研究联合应用多个DNA位点在尸源鉴定方面的应用价值。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的方法通过对无名尸体的有关检材与可疑双亲或子女进行亲权鉴定。结果　在 80例刑事案件尸源鉴定中 ,38例无名尸体采用较新鲜肌肉 ,通过扩增VNTR、STR多个位点得以判明尸源 ;2 9例采用腐败肌肉、 6例采用骨骼和 2例采用牙齿 ,通过扩增多个STR位点判明了尸源。仅有 1例采用腐败肌肉、 2例采用骨骼未能确定尸源。结论　运用多个位点进行亲权方法可以准确地判明尸源鉴定 ,特别对高度腐败尸体、尸块不全等情况下的尸源鉴定具有很高的实用价值     

法医SNP复合检测体系的构建及应用

目的构建48-SNP位点复合检测体系,用于个体识别、性别鉴定、ABO基因分型。方法采集225份无关个体样本(血斑及口腔拭子),18份案例样本(不同组织及体液斑),选择43个常染色体位点、4个ABO基因位点和1个性别鉴定位点,根据单碱基延伸技术通过GenomeLabTMSNPstream基因分型系统进行SNP分型;并检测体系灵敏度、同一个体不同组织同一性及模拟腐败检材。结果 48-SNP体系分型结果与测序结果的一致性为100%,最小DNA检出量为0.25ng,不同组织来源样本检测同一性很好;利用该体系检测225名无关汉族个体,所有位点均符合Hardy-Weinberg平衡,整个系统的随机匹配概率为9.4×10-18,累积非父排除率(CEP)为0.999 788,累积个体识别率大于0.999 999 999 999 999 99。结论本文48-SNP体系能同时进行个体识别、ABO基因分型和性别鉴定,可以作为现有STR检验体系的补充。     

Goldeneye~(TM) DNA身份鉴定系统22NC试剂盒的法医遗传学调查

目的调查Goldeneye TM DNA身份鉴定系统22NC试剂盒中所包含的21个常染色体STR基因座在汉族人群中的遗传学数据,并考察该试剂盒的法医学应用价值。方法应用Goldeneye TM DNA身份鉴定系统22NC试剂盒对华东地区500名汉族健康无关个体进行常染色体STR基因座分型检测,统计分析21个常染色体STR基因座的频率数据、群体遗传学参数及连锁不平衡状况。结果 21个常染色体STR基因座在华东汉族人群中均符合Hardy-Weinberg平衡,各基因座之间相互独立。DP值均大于0.85,在群体中的CDP值为1-3.616 5×10-26,二联体累积非父排除率为1-2.786 81×10-6,三联体累积非父排除率为1-8.545 82×10-10。结论Goldeneye TM DNA身份鉴定系统22NC试剂盒中所包含的21个常染色体STR基因座在华东汉族人群中具有良好的多态性,联合Goldeneye TM DNA身份鉴定系统20A可以满足双亲皆无的全同胞鉴定要求,为该类案件的解决提供便利的工具。     

焦磷酸测序分析短片段牙釉质蛋白基因进行性别鉴定

目的采用焦磷酸测序技术分析短片段牙釉质蛋白基因进行性别鉴定并用于骨骼及腐败生物检材的检测。方法应用blast软件,确定牙釉质蛋白基因(Amel)上1段含有3个SNP位点及1个插入/缺失(indel)位点的序列作为待测靶序列,设计引物,扩增该段序列,应用焦磷酸测序技术分析扩增序列,进行性别鉴定。对方法进行准确性、灵敏度、种属特异性的测试,并用于对骨骼和高度降解DNA的检测。结果 PCR产物分别为44bp(Amel X)和45bp(Amel Y),女性测序结果为:G/G,T/T,…/…,C/C,男性测序结果为:G/T,T/A,…/C,C/A,分型图谱清晰。应用本文方法检测100份已知性别的DNA样本,结果均正确无误,方法最低DNA模板量为0.5ng,具有较好的人类种属特异性。用于高度降解DNA分析,较IdentifilerTM试剂盒具有更高的成功率且骨骼样本也得到清晰的分型结果。结论本文采用焦磷酸测序技术分析Amel的方法在法医学性别鉴定中有较好的应用价值。     

荧光标记STR分型技术检验腐败组织基因型

探讨腐败组织荧光标记STR分型检测技术的应用价值。应用含12个STR基因座及一个性别基因座的2个荧光标记的复合扩增系统,对40例1～6周的腐败肌肉提取的DNA进行扩增,用变性聚丙烯酰胺凝胶电泳,PE377测序仪分析基因型。所检测样本在12个STR基因座均扩增出特异性谱带,并可判定其基因型。荧光标记STR检测技术对腐败组织分型可靠,在实际检案中具有较高的应用价值。     

二组分混合DNA样品STR图谱解释

对混合样品STR图谱的结果进行解释。实验模拟二组分DNA混合样品 ,复合扩增荧光检测 10个基因座 ,比较混合样品谱带 ,计算等位基因峰面积比。结果发现 :二组分DNA混合样品的等位基因数增加 ,样品的混合比例不同就出现峰面积的不平衡。在等位基因峰面积比值与样品组分混合比例接近时 ,可由峰面积比值推断混合样品的混合比例。在混合比例为 1∶2 0时 ,基本上检测不到来自少量混合成分的等位基因 ,表现为单一组分图谱 ;在混合比例为 1∶10时 ,含量低的组分的等位基因峰面积接近与主要组分的“Stutter”峰面积 ,与来自主要组分的等位基因峰面积差异很明显。能检出混合样品中少量成分等位基因的最高混合比例为 1∶10     

Validation of short tandem repeats (STRs) for forensic usage: performance testing of fluorescent multiplex STR systems and analysis of authentic and simulated forensic samples

The amplification and typing conditions for the 13 core CODIS loci and their forensic applicability were evaluated. These loci are CSF1PO, FGA, TH01, TPOX, vWA, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, and D21S11. Results were obtained using the multiplex STR systems AmpFlSTR Profiler Plus and AmpFlSTR COfiler (Applied Biosystems, Foster City, CA), GenePrint PowerPlex (Promega Corporation, Madison, WI), and subsets of these kits. For detection of fluorescently labeled amplified products, the ABI Prism 310 Genetic Analyzer, the ABI Prism 377 DNA Sequencer, the FMBIO II Fluorescent Imaging Device, and the Fluorlmager were utilized. The following studies were conducted: (a) evaluation of PCR parameter ranges required for adequate performance in multiplex amplification of STR loci, (b) determination of the sensitivity of detection of the systems, (c) characterization of non-allelic PCR products, (d) evaluation of heterozygous peak intensities, (e) determination of the relative level of stutter per locus, (f) determination of stochastic PCR thresholds, (g) analysis of previously typed case samples, environmentally insulted samples, and body fluid samples deposited on various substrates, and (h) detection of components of mixed DNA samples. The data demonstrate that the commercially available multiplex kits can be used to amplify and type STR loci successfully from DNA derived from human biological specimens. There was no evidence of false positive or false negative results and no substantial evidence of preferential amplification within a locus. Although at times general balance among loci labeled with the same fluorophore was not observed, the results obtained were still valid and robust. Suggested criteria are provided for determining whether a sample is derived from a single source or from more than one contributor. These criteria entail the following: (a) the number of peaks at a locus, (b) the relative height of stutter products, and (c) peak height ratios. Stochastic threshold levels and the efficiency of non-templated nucleotide addition should be considered when evaluating the presence of mixtures or low quantity DNA samples. Guidelines, not standards, for interpretation should be developed to interpret STR profiles in cases, because there will be instances in which the standards may not apply. These instances include (a) a primer binding site variant for one allele at a given locus, (b) unusually high stutter product, (c) gene duplication, and (d) translocation.     

中国“罪犯DNA数据库”STR基因座研究

选择合适的 STR基因座 ,建立中国“罪犯 DNA数据库”模式库。以 2 2 11名汉族、15 0名维吾尔族、10 4名回族群体为分析对象 ,提取罪犯血样 DNA,用复合 PCR和四色荧光技术检测了 D3S135 8、v WA、FGA、D8S1179、D2 1S11、D18S5 1、D5 S818、D13S317、D16 S5 39、TH0 1、TPOX、CSF1PO、D7S82 0等 13个 STR基因座及性别 Amelogenin基因座。在 13个 STR基因座中 ,除 TH0 1、TPOX基因座外 ,其余各基因座的个体鉴别能力 (DP)值均接近 0 .9,杂合度(H)均大于 0 .7,排除概率 (PE)大都在 0 .5以上 ,其中 FGA、D8S1179、D2 1S11和 D18S5 1基因座 DP≥ 0 .95 ,H≥ 0 .85 ,PE≥ 0 .6 5 ,表明它们在法医学上极有应用价值。TH0 1和 TPOX在多态性方面较差 (H分别为 0 .6 430和 0 .6 2 96 ,PE分别为0 .40 46和 0 .370 1) ,但也符合应用于法医学的要求。13个基因座的平均偶合率为 5× 10 - 1 5 ～ 1.2× l0 - 1 4 ,适合作为中国人群的遗传学标志 ,用于建立中国“罪犯 DNA数据库”     

High‐Throughput STR Analysis for DNA Database Using Direct PCR,

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high‐throughput and cost‐effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter‐loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time‐ and cost‐saving manner.     

DNA Purification Cell Lysis and Wash Step Modifications for Low-Template DNA Sample Processing,

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low-template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re-extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low-template DNA sample processing are not enough to justify the risk associated with additional manipulation.     

Constructing universal multiplex PCR systems for comparative genotyping

Analysis of length polymorphisms at STR loci in the human genome has become a standard approach for comparative genotyping in many areas including disease research and diagnostics, parentage assessment, investigations of human diversity, and forensic science. The simultaneous analysis of multiple STR loci through multiplex PCR and multicolor fluorescence detection offers sample conservation, high throughput, and automated genetic analysis. Careful design and optimization of tetranucleotide STR multiplexes has led to reliable, standardized systems that powerfully differentiate and distinguish individual human DNA profiles. The development of these multiplex systems involved a rigorous experimental strategy that included careful selection of PCR primer sequences (for yield, specificity, and multiplex compatability), along with optimization of PCR component concentrations, thermal cycling parameters, and fluorescence detection conditions. This developmental approach rendered well-characterized DNA typing systems that are high performing (sensitive, specific, and balanced), optimized to universal parameters (same reaction conditions), resilient to fluctuations in reaction conditions, and simple to implement and use routinely.     

A genetic basis for anomalous band patterns encountered during DNA STR profiling

Since 1995 the Forensic Science Service (FSS) has carried out DNA profiling of reference samples for the UK National DNA Database and in forensic casework using two multiplex STR profiling systems. During this period, profiles with anomalous banding patterns, although comparatively rare, have been encountered regularly. The FSS has collected instances of triallelic patterns and aberrant diallelic patterns. A systematic examination of these patterns has provided insight into their underlying genetic cause. The triallelic patterns could be classified into two types based on the relative intensities of their component alleles. In the Type 1 pattern the alleles were of uneven intensity, whereas in the Type 2 pattern, all three alleles were of even intensity. Evidence is presented that the more frequent Type 1 pattern is the result of somatic mutation at a heterozygous locus, and the Type 2 pattern is the result of a localized chromosomal rearrangement at a heterozygous locus. Directly from the Type 1 pattern, it was possible to deduce the size difference between the progenitor and mutated allele. All mutational changes were found to be multiples of four nucleotides, suggesting the loss or addition of one or more tetrameric repeat units. Aberrant diallelic patterns were identified by analysts due to an unexpectedly large difference in intensity between alleles at a heterozygous locus. While some of these diallelic patterns are likely caused by the same genetic phenomena described above occurring at a homozygous locus, others are demonstrated to be caused by a mutation in the primer binding sequence, leading to a reduction in amplification efficiency of one allele. It is concluded that based on a visual inspection of a profile, it is possible to infer a likely genetic basis directly from the triallelic pattern. By contrast, the aberrant diallelic patterns can be due to any one of a number of possible genetic effects.     

Methamphetamine impurity profiling using a 0.32 mm i.d. nonpolar capillary column

Classification of seized methamphetamine by impurity profiling can provide very useful information in criminal investigations of drug traffic routes, sources of supply and relationships between seizures. The aim of this study is to improve and develop an analytical method for detecting impurities such as starting materials and by-products in illegally prepared methamphetamine.HCl samples. A 50mg sample of methamphetamine.HCl was dissolved in 1 ml of buffer solution (four parts 0.1M phosphate buffer pH 7.0 and one part 10% Na2CO3). Impurities were extracted with 0.5 ml of ethyl acetate containing four internal standards (ISs) (n-decane, n-pentadecane, n-nonadecane and n-hexacosane) and analyzed by gas chromatography (GC) using a flame ionization detector (FID) on a DB-5 capillary column (0.32 mmi.d. x 30 m, film thickness 1.0 microm). The use of a middle-bore column offered better separation of the impurity peaks. The correction of the retention times of impurity peaks with four ISs made peak identification very accurate for subsequent data processing. Twenty-four characteristic peaks were selected for comparison and similarity and/or dissimilarity between samples, and the data were evaluated by the Euclidean distance of the relative peak areas after logarithmic transformation. The results indicate that the present method would be useful for methamphetamine impurity profiling.     

Accurate STR allele designations at the FGA and vWA loci despite primer site polymorphisms

Polymerase chain reaction (PCR)-based STR DNA typing systems are used extensively in the field of human identification. Under optimal PCR conditions, the amplicon yield from both alleles of an STR locus is expected to be approximately equivalent. However, it is reasonable to expect that rare genomic sequence polymorphisms will co-localize with well-designed primer sets and induce allele imbalance or "dropouts". Two samples were identified in the course of genotyping thousands of individuals with AmpF/STR Profiler Plus that showed strong disparity in amplitude peak height of heterozygous peaks at the loci vWA and FGA. These samples were reamplified at reduced annealing temperature in an attempt to balance the peak heights. Nucleotide sequencing documented polymorphisms at the PCR primer binding sites of the affected alleles. The results indicate that reducing the annealing temperature to improve primer-binding efficiency at the mismatch and employing an alternative multiplex enhanced the data from both samples. Reducing annealing temperatures could provide a simple general solution to improving data quality for samples where polymorphisms are suspected to cause allele imbalance. Finally, we report on additional polymorphisms surrounding the vWA locus in a genetically diverse population.     

广东地区汉族人群 13个 STR基因座的频率调查

目的调查广东地区汉族无关个体的 13个 STR基因座（ D3S1358、 vWA、 FGA、 D8S1179、 D21S11、 D18S51、 D5S818、 D13S317、 D7S820、 D1 6S539、 TH01、 TPOX、 CSF1PO）多态性,研究其在法医学检验中的应用价值。方法用 AmpFlSTR Profiler Plus及 Cofiler二个荧光标记系统对新鲜血样进行 13个基因座的复合扩增,用 ABI 377-96全自动测序仪对扩增产物进行检测,用 GenoTyper软件进行基因分型。结果 13个基因座 PIC >0.5,DP >0.76,家系调查符合孟德尔遗传规律。结论含有 13个 STR基因座的二个荧光标记复合扩增系统可满足法医物证学的个体识别及亲权鉴定的需要。