微量检材DNA提取方法

法医DNA鉴定中常见的微量检材,是指含有微量核DNA的降解检材(例如陈旧骨骼等)和附着载体上生物组织量少的检材。为了获得“大量”的检材DNA就必须增加检材用量,扩大反应体系。应用大反应体系提取微量检材DNA时,常出现提取溶液DNA含量过低,而不能获得DNA沉淀。如果不采用有效的方法,就会由于提取操作不当而引起DNA损失,导致PCR反应失败,直接影响案件的侦破和法庭证据的科学性。本文应用正丁醇浓缩法成功地解决了微量检材DNA的提取问题,在实际的案件鉴定中取得了满意的结果。1材料与方法1.1微量检材所用微量检材均来自案件鉴定所用…     

一种改良的骨组织DNA提取方法

骨组织耐腐败,当其他软组织已完全腐败后,只能用骨组织进行种属鉴定,性别鉴定和个人识别.骨组织DNA提取是骨组织DNA分析的关键.作者曾用多种方法提取过骨组织DNA,本文报道一种改良的骨组织DNA提取方法.     

尿液中DNA的提取与检验

尿液中DNA的STR分型检验有一定的应用价值:国外文献报道过应用于运动员兴奋剂控制^[1]中可疑尿检样品的个体识别,国内则偶有案发现场提取到尿液、尿冰、尿斑并成功进行个体识别的案例报道。与血液、精斑、唾液等检材相比,尿液STR分型的检出率较低,主要是与尿液中的成份有关。本文重点分析人类尿液成分,分类总结目前可用于提取尿液中DNA的多种方法,分析可能影响尿液STR分型检验结果的因素。     

改良醋酸铵盐析法提取福尔马林固定石蜡包埋组织DNA

目的采用改良醋酸铵盐析法提取福尔马林固定石蜡包埋组织DNA,并评价其应用价值。方法取死后24h内人体肾组织用10%中性福尔马林溶液固定7d,石蜡包埋。采用酚/氯仿法、Chelex-100法及改良醋酸铵盐析法提取DNA。经用紫外分光光度计法、AGE及PCR-PAGE检测比较不同方法提取DNA的质量。结果改良醋酸铵盐析法、酚/氯仿法、Chelex-100法OD260/OD280比值分别为1.962±0.195、2.110±0.470、1.018±0.124,两两比较均有显著性差异(P<0.05);3种方法提取DNA含量(μg)分别为0.515±0.447、0.328±0.345、5.346±1.994;AGE电泳图谱可印证上述结果;PCR-PAGE检测显示改良醋酸铵盐析法提取DNA的谱带清晰度好于其它两种方法。结论改良醋酸铵盐析法更适合微量福尔马林固定石蜡包埋组织样本DNA的提取。     

应用磁珠法提取陈旧骨骼DNA

目的探讨采用磁珠法提取陈旧骨骼DNA的可行性。方法取经土埋或室外暴露下存放1~5年不等的10根长骨,经水洗、刮净,钻取骨密质骨粉3g,应用EQ1000磁珠试剂盒提取DNA,经复合扩增,ABI 3130XL基因分析仪电泳分离,进行STR分型检测。结果 10根长骨均获得完整的STR分型,电泳图谱基线干净,除个别大片段基因座外,等位基因荧光信号分布均衡性较好。结论采用磁珠法提取陈旧骨骼的DNA,能满足分型要求,可在实际检案中选用。     

冰冻尿液的DNA提取及其检验

呼伦贝尔地区纬度高冬季漫长、气候寒冷，年平均气温-2℃，局部最低温度达到-50℃。在这种气候条件下，案件现场勘查中，有时会发现犯罪嫌疑人排泄在雪堆上的尿液，在低温条件下形成尿冰，成为能进行DNA检验的生物物证，为案件侦破提供有力线索。本文就尿冰的DNA提取方法及STR检验进行了探讨，现报道如下。     

67例皮肤接触样本的采集及DNA提取方法的比较分析

<正>DNA分型检验已广泛应用于法医学实践检案,尤其皮肤脱落细胞DNA分型检验,对侦查破案有重要意义[1]。而对于此类检材,选择检材预处理及DNA提取方法至关重要。本文针对常见的皮肤接     

磁珠DNA自动提取系统在法医检验中的应用

磁珠DNA自动提取系统是将磁珠分离技术与DNA自动提取工作站结合运用,以达到高效提取DNA的目的,它具有操作时间短、准确性高、检材用量少等特点,特别适用于大批量生物检材的快速提取,是目前主流的DNA自动化提取方法。本文对磁珠DNA自动提取系统的基本组成、工作原理、操作流程及其在法医检验领域中的应用等进行了综述,并对该系统的发展前景进行了展望。     

骨松质的DNA提取方法应用

骨骼的STR检验作为尸骸个体识别的有效手段,在法庭科学中应用广泛.在工作中,经常遇到案件中只剩残肢的尸骸,无法辨认,给案件的定性带来很大难度.尸源的快速个体识别对案件定性至关重要,目前此类案件检验一般进行骨密质的DNA检验,但具有相对检验难度大、操作复杂等特点,而骨松质和骨密质均属骨组织,骨松质结构特殊,DNA含量较多...     

用二步消化法提取指甲DNA

<正>在日常检案工作中，对腐败尸体的指甲进行DNA检验，扩增效果有时不稳定，特别是埋于泥土、弃于粪坑、污水等环境中的指甲检材，常需多次反复处理才能得到满意DNA检测结果。究其原因，可能与没有得到有效的DNA模板有关。作者采用“二步消化法”，可以去除表层降解DNA，减少PCR抑制物，从而可有效地提高了DNA模板质量，提高了指甲DNA的检验成功率。     

应用自动化工作站提取常见生物样本DNA

目的建立使用自动化工作站提取法医案件生物样本DNA的方法。方法选用Biomek 3000自动化工作站,采用DNA IQTM系统及Chelex法对法医案件中常见生物样本进行DNA提取,荧光定量技术进行定量,PCR扩增16个STR基因座并与手工提取方法比较。结果与手工提取方法进行比较,选用自动化工作站结合使用DNAIQTM系统及Chelex法提取DNA可获得满意的STR检验结果。结论自动化工作站可用于法医案件中常见生物样本的DNA提取。     

DNA提取方法和PCR循环次数对STR扩增成功率的影响

目的探讨常见载体上的微量血痕DNA的提取方法、PCR循环次数对STR扩增成功率的影响。方法分别应用Chelex-100法及Chelex-100结合纯化法对8种载体上不同大小的血痕样本进行DNA提取,并采用28次、30次及34次PCR循环进行STR扩增,分别观察其扩增成功率。结果经28次、30次及34次PCR循环,Chelex-100法提取DNA后的STR扩增成功率分别为0.2917,0.3333,0.4583,Chelex-100结合纯化法的STR扩增成功率分别为0.3750,0.4583,0.8750。结论用Chelex-100结合纯化法提取DNA,用34次循环扩增可提高STR基因座的检测成功率。     

粪便DNA提取及检验

目的　研究人类粪便DNA的提取和检验方法。方法　 8人份粪便样本 ,磁珠法提取DNA后 ,进行STR复合扩增和mtDNAHVI区测序分析。结果　用 2种方法提取的粪便DNA ,STR复合扩增检验均未获成功 ;方法1提取的粪便DNA有 6个样本、方法 2有 7个样本获得了清晰可读的mtDNAHVI区序列 ,并与唾液对照样本DNA的序列完全一致。结论　用本文建立的方法提取粪便DNA ,不适于STR分析 ,可通过mtDNA测序分析进行检验。     

Evaluation of five DNA extraction systems for recovery of DNA from bone

Five DNA extraction systems were assessed for their DNA extraction efficiency on samples of fresh pig bone. Four commercially available silica-based extraction kits (ChargeSwitch^® gDNA Plant Kit (Life Technologies), DNA IQ^TM System Kit (Promega), DNeasy^® Blood & Tissue Kit (Qiagen) and PrepFiler^® BTA Forensic DNA Extraction Kit (Life Technologies)) and a conventional phenol-chloroform method were tested in this study. Extracted DNA samples were quantitated with GoTaq^® qPCR Master Mix (Promega) using an Applied Biosystems^® 7500 Real-Time PCR System and the extracts were amplified using an in-house multiplex system. The phenol-chloroform extraction produced higher yields of DNA than the silica-based extraction methods. Among the silica-based extractions ChargeSwitch^® gDNA Plant Kit recovered the highest amounts of DNA. However, all methods produced DNA that could be amplified and none of the extracts contained any detectable inhibition.     

Efficiency of Casework Direct Kit for extraction of touch DNA samples obtained from cars steering wheels

Analysis of STR profiles obtained from touch DNA has been very useful to the elucidation of crimes. Extraction method may be determinant for the recovery of genetic material collected from different surfaces. Vehicle theft is one of the most common crimes in São Paulo city, Brazil, but collection of biological traces in car steering wheels is not considered, because of the belief that profiles generated won’t be able to identify the thief, only the owner. This study aimed to analyze the efficacy of extraction methods for obtaining DNA profiles in samples collected from steering wheels. Eight criminal acts were simulated with 2 different individuals each (mixture of victim and thief), in duplicate, in order to compare two extraction methods: DNA IQ™ and Casework Direct Kit (both Promega Corporation). Genetic material was collected by double swab method and quantified by Quantifiler™Trio (ThermoFisher Scientific). Amplification was conducted with PowerPlex® Fusion System (Promega). It was possible to obtain STR profiles for all experiments. The mixtures were compared with reference profiles to evaluated how many alleles of each donor were observed. Samples extracted with Casework Direct Kit obtained STR profiles with higher averages of alleles for primary and secondary donors (88.7% and 59.9%, respectively) than those extracted with DNA IQ™ (60.4% and 38.1%, respectively). This could be explained by the differences established in the protocols of both methods, since DNA IQ™ is based on successive washes and can result in loss of DNA, whereas Casework Direct Kit minimizes this problem. We concluded that Casework Direct Kit was more efficient for processing touch DNA samples than DNA IQ™.     

FTA-DNA直接提取法的研究与应用

目的建立一种简约且易于自动化操作的Whatman FTA卡血样DNA提取方法。方法取直径1．2mm FTA卡血样，分别用FTA-DNA直接提取法及FTA常规提取方法提取DNA，AB-Identifiler^TM试剂盒分别进行10山和25山体系检测比较，并筛选批量grA卡血样DNA自动化提取程序。结果200份grA卡血样分别采用2种方法提取DNA模板，25μl体系PCR-STR检测，分型结果均良好。10μl体系检测，FTA-DNA直接提取法优于FTA常规提取方法，图谱RFU值为1000～2000，采用自动化工作站运行该提取程序较手工操作DNA分型图谱均衡性更佳；采用FTA常规提取方法检测，图谱RFU值100～2000，小片段优先扩增现象明显，且有19％的样本出现丢峰现象，采用自动化工作站运行该程序对其结果改善不明显。经工作站运行FTA-DNA直接提取法自动程序及10山体系检测本2万余份FTA卡采集的血样，均获得16个STR基因座DNA分型结果。结论本文建立的FTA-DNA直接提取法适用于FTA卡血样自动化DNA建库。     

改良IPEP法用于痕量DNA检测的实验研究

目的探讨改良扩增前引物延伸(IPEP)法对痕量DNA样本STR检测分型的效果。方法用改良IPEP法对痕量样品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AmpFLSTR~ Indentifiler~试剂盒作基因型检测。结果该方法可增加模板DNA约200~1100倍。基因组DNA不低于0.025ng时,可获得15个STR基因座和Amelogenin性别基因座的分型结果。基因组DNA0.01~0.025ng时,可获得9个以上基因座的分型结果。结论改良IPEP法可有效提高痕量DNA样本STR分型检验的灵敏度,有较好的实用价值。     

中国法庭科学DNA数据库

本文综述了中国法庭科学DNA数据库的建立和发展过程、DNA数据库结构、内容、特点、作用以及存在的问题、发展方向、展望,目的是为如何进一步建设好具有中国特色的DNA数据库提供借鉴。     

The NucleoSpin DNA Clean-up XS kit for the concentration and purification of genomic DNA extracts: An alternative to microdialysis filtration

Traditionally, DNA extracts from biological evidence items have been concentrated and rinsed using microdialysis filtration units, including the Centricon^® and Microcon^® centrifugal filter devices. As an alternative to microdialysis filtration, we present an optimized method for using NucleoSpin^® XS silica columns to concentrate and clean-up aqueous extracts from the organic extraction of DNA from biological samples. The method can be used with standard organic extraction and dithiothreitol (DTT)-based differential extraction methods with no modifications to these methods prior to the concentration and clean-up step. Extracts from laboratory-prepared bloodstains, saliva and semen stains have been successfully amplified with both qPCR and STR assays. Finally, the total time to process a set of samples with the NucleoSpin^® XS column is approximately 30 min vs. approximately 1.5 h with the Centricon^® YM-100 filter device.     

人线粒体DNA序列分析在法医学中的应用研究及其进展

综述人线粒体DNA(m tDNA)序列分析在法医学种属鉴别、个体识别,以及个体年龄推断中的应用研究及其进展,展望对m tDNA异质性的研究及建立人m tDNA数据库,并具有重要的法医学实践意义。