不同前处理方法提取牙齿DNA的比较

目的对三种不同前处理方法提取的牙齿DNA浓度进行比较,建立一种操作简便、经济适用、浓度较高的牙齿DNA提取的前处理方法。方法选择源自7具尸体的共21颗磨牙,每具尸体的3颗磨牙随机按牙屑法、球磨法、液氮研磨法进行前处理,并分别称取50 mg,采用Auto Mate Express~(TM)法医DNA提取系统提取DNA,对三种方法提取的DNA浓度和STR分型结果进行比较。结果牙屑法、球磨法和液氮研磨法提取的DNA质量浓度分别为0.055 6~1.989 1、0.036 6~1.175 6和0.037 8~1.249 0 ng/μL,牙屑法提取的DNA质量浓度较高(P0.05),且STR分型成功率高。结论牙屑法结合Auto Mate Express~(TM)法医DNA提取系统是提取牙齿DNA的一种切实可行的方法,可应用于法医学鉴定实践中。     

利用AutoMate Express~(TM)自动化法医DNA提取系统提取骨骼及牙齿DNA

目的探讨建立骨骼及牙齿DNA自动化提取的新方法。方法将33份骨骼及15份牙齿样本分别用冷冻研磨和手工处理两种方法研磨成粉,采用AutoMate ExpressTM自动化法医DNA提取系统提取DNA并定量。结果 AutoMate ExpressTM自动化法医DNA提取系统能够在3h左右完成骨骼、牙齿DNA的提取,两种方法处理的骨骼样本所得DNA质量浓度差异无统计学意义。冷冻研磨处理的骨骼和牙齿样本均获得了较好的STR分型结果,且牙齿样本所得DNA质量浓度高于手工提取所得。结论应用AutoMate ExpressTM自动化法医DNA提取系统是自动化提取骨骼、牙齿DNA的一种新方法,可应用于法医实际案件检验。     

3种骨骼DNA提取方法的应用效果比较

目的对3种方法提取骨骼DNA的效果进行比较,为实际应用中选择方法提供参考。方法应用骨骼孵化液法、DNA Investigator试剂盒法和CTAB法对同一骨骼样本进行脱钙、消化、提取DNA,用紫外分光光度计检测DNA的浓度值;使用Identifilerplus试剂盒进行PCR扩增,3130xl型遗传分析仪检测分型,并用SPSS 19.0软件对各项实验结果进行统计分析。结果 1g骨粉样本经上述3种方法提取,得到的DNA浓度分别为26.53ng/μL±5.47ng/μL、23.63ng/μL±4.56ng/μL、14.93ng/μL±3.88ng/μL;单因素方差分析表明3组数据之间差异性具有统计学意义。PCR扩增后电泳检测结果显示,骨骼孵化液法和DNA Investigator试剂盒法基因座检出率和峰值大致相同,均优于CTAB法。结论本文比较的3种方法均可用于骨骼样本的实际检案,检出率较高的两种方法可作为优选方案。     

人体脂肪组织DNA提取方法的比较

在法医物证学检验中，进行DNA分型的检材多为血液（痕）、毛发、指甲、肌肉组织及骨骼等，极少选择脂肪组织。脂肪组织采用常规方法提取模板DNA效果不佳，本文通过对不同提取方法进行比较，以探索可适用于脂肪组织DNA提取的方法。     

两种提取骨骼、牙齿DNA的方法

目的建立提取骨骼、牙齿DNA的新方法。方法收集380例骨骼、牙齿检材,其中347份为常规骨骼、牙齿(常规组),33份为陈旧骨骼、牙齿(疑难组)。常规组检材以Handy-Eco仪器、疑难组则用冷冻研磨仪研磨成粉,以Kingfisher自动化系统提取DNA,用Identifiler Plus进行STR分型检测。结果用HandyEco Kingfisher法(H-K法)成功提取345例常规骨骼、牙齿检材的DNA,成功率99.42%;用Freeze-mill Kingfisher法(FM-K法)成功获取32例疑难骨骼、牙齿检材的DNA,成功率96.97%。结论采用H-K法和FM-K法对骨骼和牙齿DNA的检验成功率较高,可选择应用于工作实践中。     

牙齿的DNA提取及STR分型研究

目的建立有效的牙齿DNA提取方法。方法使用物理及化学方法去除牙齿表面污染物,经脱钙、裂解、纯化从牙粉中提取DNA进行STR分型。结果对96例牙齿检材进行DNA检验,获得STR分型的有94例,在查找尸源的案件中发挥了重要作用。结论本方法操作简单快速,能够显著提高牙齿DNA检验的成功率。     

2种陈旧骨骼、牙齿DNA纯化方法的比较

目的比较有机法+QIAquick纯化法和DNA IQ磁珠法对陈旧骨骼和牙齿DNA的纯化效果。方法选择10份陈旧骨骼和12份牙齿样本,进行消化后分别采用有机法+QIAquick纯化法和DNA IQ磁珠法进行提取纯化,进行DNA定量后用SinofilerTM试剂盒进行检测。结果 2种方法纯化的骨骼、牙齿DNA的IPC CT值无显著差异。有机法+QIAquick纯化法纯化的骨骼、牙齿DNA平均浓度分别为0.180ng/μL±0.068ng/μL和0.132ng/μL±0.027ng/μL,所有样品均获得全部基因分型。DNA IQ磁珠法纯化的DNA平均浓度分别为0.038ng/μL±0.028ng/μL和0.036ng/μL±0.007ng/μL,有5份骨骼和6份牙齿样本仅获得部分基因分型或未能分型。结论有机法+QIAquick纯化法对陈旧骨骼、牙齿DNA的纯化效果优于DNA IQ磁珠法。     

陈旧骨骼DNA提取方法的应用研究

在法医实践中,DNA检验已成为个体识别鉴定的常规技术。现场提取的检材中,骨组织具有耐腐败特性,当其他检材完全腐败后,骨组织仍能够作为DNA检验的重要材料。但陈旧尸骨骨组织中DNA含量少且高度降解,给提取DNA带来一定难度。本文采用较高温度脱钙,在磷酸盐缓冲液条件下经SDS-PK、GuSCN消化裂解骨细胞,含DNA的裂解产物经硅珠纯化,从1~16年的陈旧骨骼中提取到高质量DNA模板用于STR分型,成功率为95.5%。现将方法报告如下:1材料与方法1.1材料骨骼样本89根,来源于送检案件。其中完整长骨78根,断骨和骨片11根。现场条件有泥土掩埋、水…     

纳米磁珠提取法在骨骼DNA提取中的应用

目的对纳米磁珠法提取纯化骨骼DNA的效果进行比较评价,为方法选择提供应用参考。方法取泥土掩埋、水中浸泡1~10年不等的25根长骨,经水洗、刮净,液氮冷冻研磨器将骨骼研磨成粉末状,分别应用纳米磁珠提取法和King Fisher仪器自动化提取法提取DNA,IdentifilerPlus试剂盒进行扩增,ABI 3100遗传分析仪进行STR分型检测;对两种方法提取的DNA定量和经扩增、分型检测的结果进行比较。结果骨骼样本采用纳米磁珠法提取到的样本DNA(1.237 5ng/μL±0.319 2ng/μL),较之King Fisher法的浓度(0.506 2ng/μL±0.280 5ng/μL)更高,两种方法间差异具有统计学意义(P0.05);而纳米磁珠法的分型成功率亦更高,两种方法间差异具有统计学意义(P0.05)。结论用纳米磁珠提取纯化骨骼DNA,能得到高质量DNA模板,有利于提高分型检验的成功率,可在实际检案中选择使用。     

利用AutoMate Express~(TM)系统提取陈旧性骨骼DNA

目的建立利用AutoMate Express~(TM)系统提取陈旧性骨骼DNA的方法。方法将骨骼用冷冻研磨机研磨成骨粉,经AutoMate Express~(TM)系统提取后,用Identifiler~Plus、MiniFiler~(TM)试剂盒扩增分型。结果10例保存在不同环境中、死亡时间在10~20年的骨骼样本利用AutoMate Express~(TM)系统3 h内完成DNA提取,有8例获得完整STR分型。结论 AutoMate Express~(TM)系统能快速、高效地提取陈旧性骨骼DNA,可应用于法医实际案件检验。     

A Simple and Efficient Method of Extracting DNA from Aged Bones and Teeth

DNA is often difficult to extract from old bones and teeth due to low levels of DNA and high levels of degradation. This study established a simple yet efficient method for extracting DNA from 20 aged bones and teeth (approximately 60 years old). Based on the concentration and STR typing results, the new method of DNA extraction (OM) developed in this study was compared with the PrepFiler? BTA Forensic DNA Extraction Kit (BM). The total amount of DNA extracted using the OM method was not significantly different from that extracted using the commercial kit (p > 0.05). However, the number of STR loci detected was significantly higher in the samples processed using the OM method than using the BM method (p < 0.05). This study aimed to establish a DNA extraction method for aged bones and teeth to improve the detection rate of STR typing and reduce costs compared to the BM technique.     

两种骨骼及牙齿纯化方法的比较分析

目的比较硅珠法和硅胶膜法对骨骼和牙齿的纯化效果。方法选择6根骨骼和8颗牙齿,进行消化后分别采用硅珠法与硅胶膜法进行纯化,用Global Filer~(?)试剂盒进行扩增检测,通过比较检出率和峰高来评价这两种方法。结果两种纯化方法均成功检测出了骨骼和牙齿的STR分型。同一样本中,相同基因座上两种方法检出的等位基因分型结果完全一致。两种方法处理得到的平均峰高差异无统计学意义。结论硅胶膜法在骨骼及牙齿的纯化中能满足实际常规检案的要求,且和硅珠法无明显差异,但在操作上更具优势,缺点是成本较高,在实际工作中可以选择使用。     

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.     

96孔过滤板在接触性DNA自动化检验中的应用

目的将96孔过滤板用于自动化工作站,对接触性DNA进行检验。方法收集553份现场提取的附着在烟蒂、饮料瓶、门窗把手、电源网线头、作案工具、手套上的微量接触性生物检材,在96孔过滤板上进行裂解后整体离心,分离载体与裂解液;应用自动化核酸提取纯化仪结合M48磁珠纯化试剂盒提取纯化DNA,采用IdentifilerPlus试剂盒进行扩增,3500XL测序仪电泳分型,ID-X专家系统分析结果。结果在553份检材中,成功获得STR分型的有271份,检验成功率在31.6%~97.3%之间,烟蒂、饮料瓶检材的成功率均在90%以上,提取纯化过程约90min。结论将96孔过滤板用于自动化工作站,可在实现批量接触性DNA的快速、自动化提取的同时,有效提高接触性DNA检出率。     

运用酚-氯仿法结合磁珠法提取蝇蛆体内人类DNA

目的建立运用酚-氯仿法结合磁珠法从蝇蛆嗉囊内提取人类DNA的方法,从而提高STR分型检验的灵敏度。方法采用酚-氯仿法对蝇蛆嗉囊内容物中的人类DNA进行提取,提取产物经磁珠法纯化浓缩后用QuantifilerTM人类DNA定量试剂盒在7500型实时荧光定量PCR仪上进行PCR定量,再用AmpF■STR IndentifilerTM试剂盒在3130XL-Avant遗传分析仪上对这些DNA样本进行STR分型。结果本研究建立的方法可增加模板DNA浓度约为单独使用酚-氯仿法的2倍。用此方法提取到的DNA浓度[（0.218±0.041）ng/μL]可获得全部16个STR分型结果。结论酚-氯仿法结合磁珠法可以有效地提高提取到的人类DNA样本STR分型检验的灵敏度,对于从事法医昆虫学方面研究的工作者有较好的实用价值。     

STR typing of ancient DNA extracted from hair shafts of Siberian mummies

The aim of this study was to determine if ancient hair shafts could be suitable for nuclear DNA analysis and to develop an efficient and straightforward protocol for DNA extraction and STR typing of ancient specimens. The developed method was validated on modern and forensic samples and then successfully applied on ancient hairs collected from Siberian mummies dating from the 16th to the early 19th centuries. In parallel extractions including or excluding a washing step were performed at least two times for each sample in order to evaluate the influence on the quantity of nuclear DNA yielded and on the typing efficiency. Twelve ancient individuals were analyzed through our approach and full and reliable profiles were obtained for four of them. These profiles were validated by comparison with those obtained from bone and teeth DNA extracted from the same ancient specimens. The present study demonstrates that the washing step cannot be considered as deleterious for DNA retrieval since the same results were obtained by the two approaches. This finding challenges the hypothesis that recoverable nuclear DNA is only found on the outer surface of hair shafts and provides evidence that nuclear DNA can be successfully extracted from ancient hair shafts. The method described here constitutes a promising way for non-invasive investigations in ancient DNA analysis for precious or historical samples as well as forensic casework analyses.