PCR扩增循环数与低拷贝模板DNA的STR分型

目的探讨PCR扩增循环数对低拷贝模板DNA的STR分型的影响。方法模板DNA(9947A)的不同扩增用量(含低拷贝模板量),采用ProfilerPlus试剂盒,扩增循环数分别为28、30、32、34、38次,3100型基因分析仪(ABI,美国)检测结果。结果循环数从28次增至38次,模板DNA量最低检出量可从0.25ng减少至0.0312ng;先循环28次后,每反应加0.3μlAmpliTaqGoldDNA聚合酶,再循环6次较1次34个循环的检测灵敏度高,相当于1次38个循环的效果。结论增加PCR扩增循环数,可能影响低拷贝模板DNA的STR分型。     

口腔拭子DNA检验的实时定量研究

目的寻求提高口腔拭子的DNA检验效率的方法。方法对来源于不同个体或同一个体不同擦拭次数的口腔拭子用Chelex-100或磁珠法提取的DNA用实时定量PCR技术进行定量,同时用Identifiler复合扩增系统在ABI3100遗传分析仪上对这些DNA样品进行STR分型。结果在建立的Identifiler系统8μl扩增体系中,3个月内的口腔拭子用Chelex-100法提取的DNA模板1μl量较3μl量扩增效果好,磁珠法提取的DNA模板用量大小对复合扩增检测影响较小。结论用棉签擦拭颊粘膜5次制备口腔拭子,取其头部的约1/4用200μlChelex-100法提取DNA,然后用1μl模板进行复合扩增,是提高口腔拭子的DNA检验效率的简便可行的方法,但陈旧口腔拭子用磁珠法提取更能保证复合扩增分型成功。     

Chelex-100提取生物检材DNA实时PCR定量研究

目的研究Chelex-100法提取的生物检材DNA用量与复合STR分型成功率的关系。方法113份各种生物检材采用Chelex-100法提取DNA，应用Quantifiler人类DNA定量试剂盒在ABI 7500荧光定量PCR仪上进行实时PCR定量，同时用Identifiler复合扩增系统在ABI 3100遗传分析仪上对这些DNA样品进行STR分型。结果各种生物检材提取的DNA浓度分别为：37份滤纸、纱布血痕0．042～5．28ng／μl，16份口腔拭子1．15—4．21ng／μl，18份烟头0．016～1．46ng／μl，10份肋软骨0．531—14．40ng／μl，8份肌肉5．75—24．80ng／μl，7份指甲0．788—11．50ng／μl，17份精斑0．79～99．50ng／μl。在建立的8μl扩增体系中，根据上述结果，调整用于复合STR扩增的DNA模板量在0．5—3ng之间，大部分样品可获得完全的STR分型。结论Chelex-100法提取的检材DNA模板用量在0．5—3ng之间可得到有效STR扩增，浓度为0．5ng／μl以上的DNA样品，用小体积模板（1μl）比大体积（3μl）模板扩增效果好。     

19个常染色体与4个Y染色体STR复合扩增体系的建立

目的建立19个常染色体STR及Amelogenin和4个Y染色体STR基因座复合扩增体系,并对其效能进行评估。方法用五色荧光标记20＋4Y—STR基因座,建立同步扩增检测体系,用ABI3130XL遗传分析仪对扩增产物进行电泳,GeneMapperID3．2软件进行基因分型;检测体系的灵敏度、均衡性、稳定性、特异性、同一性和稳定性,并观察混合、降解及微量检材的分型情况。结果采用本文体系,DNA模板量在0．05～1．00ng时,分型准确,均衡性、特异性好;混合、降解及微量检材分型正确。该19个常染色体STR基因座的累计个人识别率大于0．999999999,三联体累计非父排除率达0．999999985,Y—STR单倍型多态性为0．592。结论本文建立的复合扩增体系分型准确,稳定,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。     

19个常染色体与4个Y染色体STR复合扩增体系的建立

目的建立19个常染色体STR及Amelogenin和4个Y染色体STR基因座复合扩增体系,并对其效能进行评估。方法用五色荧光标记20+4Y-STR基因座,建立同步扩增检测体系,用ABI 3130XL遗传分析仪对扩增产物进行电泳,GeneMapperID 3.2软件进行基因分型;检测体系的灵敏度、均衡性、稳定性、特异性、同一性和稳定性,并观察混合、降解及微量检材的分型情况。结果采用本文体系,DNA模板量在0.05~1.00ng时,分型准确,均衡性、特异性好;混合、降解及微量检材分型正确。该19个常染色体STR基因座的累计个人识别率大于0.999 999 999,三联体累计非父排除率达0.999 999 985,Y-STR单倍型多态性为0.592。结论本文建立的复合扩增体系分型准确,稳定,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。     

安徽汉族人群21个STR基因座遗传多态性

本文对安徽汉族2 997名无关个体21个常染色体STR基因座进行遗传多态性调查。采用AGCU EX22人类荧光标记STR复合扩增检测试剂进行复合PCR扩增,用ABI3130型遗传分析仪进行毛细管电泳和基因型分析。结果在21个STR基因座共有309个等位基因和1 344种基因型;其分布符合Hardy-Weinberg平衡(P0.05)。该系统在安徽地区汉族人群中具有较好的亲权鉴定及个体识别能力。     

陈旧性骨骼DNA提取方法的探索

目的探索陈旧性骨骼DNA的提取方法。方法收集4~15年陈旧骨骼样本,去除表面污染物,经脱钙、裂解提取各样本DNA,使用QIAquickPCR Purification试剂盒进行纯化,检测DNA纯度和浓度,应用Power PlexFusion荧光标记复合扩增系统进行扩增,AB-3500型遗传分析仪检测STR分型。结果 15例陈旧性骨骼经提取、纯化,提取的DNA模板浓度较高,在42.9~176.4ng/μL之间,A260/A280值较稳定,在1.06~1.40之间;所有样本均获得完整STR分型。结论该方法简便快速,提取效果好,能够适用于法医学实际检案。     

D3S1358基因座重复序列分析1例

1案例资料1.1简要案情作者在对1例非正常死亡案件死者的血样进行DNA检验中,发现D3S1358基因座呈三带型。1.2检验方法STR分型按照硅珠法[1]提取血样模板DNA,用Identifiler Plus荧光检测试剂盒进行PCR扩增,产物在ABI 3130XL遗传分析仪进行分型检测,使用GeneMapper ID-X软件进行数据分析。筛选阳性克隆设计的引物序列见表1。     

9个犬STR基因座在犬类鉴定中的应用

目的建立犬的复合扩增体系对犬进行个体识别和亲权鉴定。方法用建立的犬复合扩增体系进行PCR反应,用ABI310型遗传分析仪对扩增产物进行检测。结果鉴定结果表明,9个犬STR基因座复合扩增体系,各个基因座扩增平衡,结果稳定。结论9个犬STR基因座复合扩增体系可以进行犬的个体识别和亲权鉴定。     

多重置换扩增技术用于法医学微量DNA检测效果

目的探讨多重置换扩增(MDA)技术对法医学微量DNA样品STR检测分型的效果。方法用MDA技术对不同模板量DNA进行全基因组扩增(WGA),扩增产物用实时荧光定量PCR技术定量、用Profiler PlusTM试剂盒检测基因型。结果该方法可对模板DNA增加104～106倍。1ng样品DNA的MDA产物可获得9个STR基因座和Amelogenin性别基因座的准确分型结果;低于0.1ng的样品DNA经MDA扩增后,基因座检出数增加,但可见等位基因不平衡或丢失现象。结论MDA技术可有效增加DNA模板量和提高微量DNA分型效果。但样品DNA量低于0.1ng时,MDA产物的STR分型结果判读须慎重。     

吸附性载体上微量血痕的DNA分型

目的研究吸附性载体上微量血痕的DNA提取及其检验。方法用Chelex-100法、QIAamp MiniKit、及QIAamp Mini Kit改良法提取吸附性载体上微量血痕中的DNA,进行PCR扩增及STR检验。结果采用Chelex-100法及QIAamp Mini Kit的分型成功率很低;采用QIAamp Mini Kit的改良法能较好的得到分型图谱。结论采用QIAamp Mini Kit的改良法能较好的提取吸附性载体上微量血痕的模板DNA。     

PCR扩增体系的体积减少对DNA分析的影响

目的探讨PCR扩增体系的体积减少对DNA分析的影响。方法4份样品采用ProfilerPlus试剂盒,在同样条件下,对50μl、25μl、12.5μl、6.25μl四种体积的体系进行扩增,扩增产物分别经ABIPRISMTM310型基因分析仪、ABIPRISMTM377型测序仪、ABIPRISMTM3100型基因分析仪电泳,并经GeneScan3.1基因扫描软件和Genotyper2.5分析软件分析得到实验结果。结果小体积的扩增体系容易出现等位基因的丢失、额外等位基因的产生等现象。结论在检材情况较差时,应该慎用小体积的扩增体系。     

低拷贝DNA模板检验方法探讨

目的探讨扩增及检测方法对低拷贝DNA模板分型检测灵敏度的影响。方法Control DNA 9947A按比例稀释,采用IdentifilerTM和DNATyper15TM试剂扩增,循环参数设置为28和28+6个循环,平行扩增3次,分别单独检测及3次合并检测,使用310及3130分析仪检测。结果28+6个循环的基因座检出率高于28个循环;等位基因不平衡及丢失与基因座没有特异性的关联,随着DNA模板量的减少,等位基因不平衡及丢失增多;将3次扩增产物混合后检测,等位基因不平衡及丢失情况减少,分型正确率增高。结论模板DNA分3次扩增后混合检测、循环数为28+6,可提高低拷贝模板基因座的检出率。     

Evaluation of a multicapillary electrophoresis instrument for mitochondrial DNA typing

Laser-induced detection of fluorescent labeled PCR products and multi-wavelength detection (i.e., multicolor analysis) enables rapid generation of mtDNA sequencing profiles. Traditionally, polyacrylamide slab gels have been used as the electrophoretic medium for mtDNA sequencing in forensic analyses. Replacement of slab gel electrophoresis with capillary electrophoresis (CE) can facilitate automation of the analytical process. Automation and high throughput can be further enhanced by using multicapillary electrophoretic systems. The use of the ABI Prism 3100 Genetic Analyzer (ABI 3100, Applied Biosystems, Foster City, CA) as well as the ABI Prism 310 Genetic Analyzer (ABI 310, Applied Biosystems, Foster City, CA) were evaluated for mtDNA sequencing capabilities and compared with sequencing results obtained on the platform currently in use in the FBI Laboratory (the ABI Prism 377 DNA Sequencer, ABI 377, Applied Biosystems, Foster City, CA). Various studies were performed to assess the utility of the ABI 3100, as well as the ABI 310 for mtDNA sequencing. The tests included: comparisons of results obtained among the ABI 3100, the ABI 310 and the ABI 377 instruments; comparisons of results obtained within and between capillary arrays; evaluation of capillary length; evaluation of sample injection time; evaluation of the resolution of mixtures/heteroplasmic samples; and evaluation of the sensitivity of detection of a minor component with reduced template on the ABI 3100. In addition, other studies were performed to improve sample preparation; these included: comparison of template suppression reagent (TSR, Applied Biosystems, Foster City, CA) versus formamide; the use of Performa DTR Gel Filtration Cartridges (Edge BioSystems Inc., Gaithersburg, MD) versus Centri-Sep Spin Columns (Princeton Separations, Adelphia, NJ) for product purification after cycle sequencing; and sample stability after denaturation. The data support that valid and reliable results can be obtained using either capillary electrophoresis instrument, and the quality of sequencing results are comparable to or better than those obtained from the ABI 377 instrument.     

Analysis of forensic samples in Banco Nacional de Datos Genéticos

Analysis of forensic samples to evaluate the rate of success for molecular markers: autosomal STRs, Y chromosome, and mitochondrial DNA. Since 2006 to date a total of 390 forensic samples were analyzed: bones, teeth, hairs, swabs, stains and paraffin embedded tissue. Bones and teeth, were pulverized in a Freezer Mill, extracted by chloroform/phenol/isoamyl alcohol method, and then purified with Centricon 100 columns. DNA from paraffin was extracted with QIAmp DNA Mini kit (QIAGEN). Mitochondrial DNA Control Region sequences were determined for regions HV1/HV2. Sequencing was performed using the BigDye^® Terminator v 1.1 Kit and analyzed in ABIPRISM^® 3100 Genetic Analyzer (AB). STRs were amplified using Amp FlSTR Identifiler^®, Minifiler^® and YFiler^® Kit (AB) and analyzed in ABI PRISM^® 3100 Genetic Analyzer and ABI PRISM^® 3130xl Genetic Analyzer (AB). Among forensic samples, bones and teeth analyzed for autosomal STRs, we obtained successful results in all of them. Incomplete typing are represented by loci of higher molecular weight, which demonstrates the poor quality of the sample due to its state of degradation and obtained better results using mini STRs. Successful results in sequencing for mitochondrial HV1 region for all samples analyzed, but in few hair samples we obtained mixed sequences and that represented important difficulties for the analysis. Age of samples and conservation are factors related which affect DNA viability. Autosomal STRs solved all the samples analyzed in our study, but Y chromosome analysis and mitochondrial DNA sequencing are also important and necessary markers in some forensic cases.     

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 a Cannabis sativa STR multiplex system for forensic analysis

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.     

探讨EOS染色剂对血迹DNA检验的影响

目的探讨被EOS染色剂处理后的血迹进行DNA检验的初步方法。方法制备EOS染色剂处理的血迹样本，分别采取纯水擦拭、75％酒精擦拭，或手术刀刮取血痕浸泡于纯水中、759／5酒精中，然后提取DNA进行下一步检测。结果采用刀刮取血痕浸泡于759／6酒精中，提取的DNA检测结果较好。结论初步实验显示，经EOS染色剂处理过的血迹，可参考刀刮取血迹置于75％酒精浸泡后提取DNA做下一步检测。     

链霉亲和素磁珠同步法检测两个miniSTR复合扩增系统

目的建立同步检测包括D5S818、D8S1179、D16S539和vWA、D21S11、D13S317基因座的两个m in iSTR系统的新方法。方法采用不同荧光染料和生物素标记引物,两个m in iSTR扩增系统在同一试管扩增,通过链霉亲和素磁珠将两个扩增系统PCR产物进行分离,用AB I 3100遗传分析仪对PCR产物检测分型。结果两个m in iSTR扩增系统可成功地进行同步扩增分型。结论应用链霉亲和素磁珠法同步检测两个m in iSTR复合扩增系统的基因型,可以降低成本,减少PCR污染,单次扩增信息量明显增高。