男性Y染色体Amelogenin Y、DYS570及DYS576缺失1例

正1案例1.1简要案情某水域发现1具高度腐败未知名尸体,法医初步判断死者为男性,提取死者肋软骨进行DNA检测。1.2检验方法使用AutoMate Express~(TM)核酸提取系统(美国Thermo Fisher Scientific公司)及配套试剂进行DNA提取,使用Power Plex~? 21系统(美国Promega公司)和GlobalFiler~(TM) PCR扩增试剂盒(美国Thermo FisherScientific公司)进行常染色体STR复合扩增。     

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。     

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PrepFiler Express~(TM)提取粪便DNA1例

<正>1案例资料1.1简要案情2015年12月,某地山区发生一起盗窃古墓案。现场勘验人员在现场发现成型大便两处,分别用多根棉签擦拭其表面后,送DNA实验室检验。1.2 DNA检验分别采用Chelex-100法、手工磁珠法(CON SUMMATE,武汉凯斯美特)、Prep Filer Express~(TM) BTA法(Prep Filer Express~(TM) BTA~(TM)法医DNA提取     

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。     

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。     

应用InnoTyper~ 21试剂盒进行毛干检验

目的探讨InnoTyper~ 21试剂盒在法医学实践中的应用价值。方法收集8名无关个体的毛发及唾液样本,采用Auto Mate Express~(TM)自动化法医DNA提取系统提取模板DNA,分别采用InnoTyper~ 21和Amp FeSTR~(TM) Identifiler~(TM) Plus试剂盒进行扩增,并对检验结果进行比较。结果采用InnoTyper~ 21试剂盒扩增时,唾液样本均可检出完整特异性分型,无毛囊毛干分型图谱峰值为57~1 219 RFU,有时可见等位基因缺失;采用Amp FeSTR~(TM) Identifiler~(TM) Plus试剂盒扩增时,唾液样本均可检出完整特异性分型,无毛囊毛干均未检出特异性片段。结论 InnoTyper~ 21试剂盒在无毛囊毛干的检案中具有一定应用价值。     

陈旧骨骼DNA提取

目的寻找从陈旧骨骼中提取DNA的有效方法。方法运用传统的有机法结合Microcon100纯化柱提取骨骼DNA。结果用常规荧光标记复合STR基因分型法可对提取到的陈旧骨骼DNA进行成功分析。结论有机法结合Micrcon100纯化柱提取陈旧骨骼DNA法可有效应用于实际检案。     

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。     

联合运用CTAB与磁珠提取陈旧骨骼DNA

<正> 用Chelex-100法或酚-氯仿法提取骨骼DNA,一般采用PK消化。该法虽对比较新鲜且有软骨组织存在的骨骼DNA提取效果较好,但对陈旧性骨骼DNA提取,难以达到理想效果。有人将CTAB(cetytrimethylammonium bromide,十六烷基三甲基溴化胺)用于陈旧性骨骼DNA提取,并采用纯化柱对DNA提取液进行浓缩纯化。由于其检验步骤相对烦琐,产物回收率受到限制。本文作者用CTAB裂解     

人骨骼及牙齿DNA提取方法的比较和优化

目的人骨骼和牙齿DNA提取方法的比较和优化。方法收集18份不同个体的长骨、30颗磨牙和同一个体2根股骨、8颗磨牙。利用TissueLyser-Ⅱ组织破碎仪和PreFiler Express BTA^TM法医DNA提取试剂盒（BTA法）,应用Automate Express^TM自动化法医DNA提取系统提取DNA,进行STR分型,与脱钙法进行比较,并进行实验条件优化。结果用TissueLyser-Ⅱ结合BTA法,约2.5h即可完成骨骼和牙齿的DNA提取,分型成功率分别为94.4%和96.7%。与脱钙法比较,两种方法获得DNA质量浓度和检出率比较接近（P〈0.05）,但BTA法在操作过程方面更具优势。最佳样本量为100mg,消化时间为2h。结论采用TissueLyser-Ⅱ组织破碎仪结合BTA法对骨骼和牙齿进行DNA提取和分型检验,能满足实际检案的要求,可在法医学实践中选择使用。     

基于二代测序平台的90个常染色体SNP分型研究

目的基于二代测序平台进行90个常染色体SNP位点分型,调查其在中国广东汉族人群中的多态性,评估其法医学应用价值。方法采集100例中国广东汉族无关个体外周血样,采用Auto Mate Express TM提取样本DNA,使用HID-Ion Ampli Seq?Identity Panel分型体系复合扩增90个SNP位点制备文库,Ion One Touch?2进行乳化PCR,Ion PGM?平台进行测序,Torrent_Suite_v4.4.2软件及HID_SNP_Genotyper_v4.3.1插件进行数据分析,计算常用法医学参数并与该群体Goldeneye TM 20A体系的检测效能进行比较。结果经Bonferroni法校正后,90个常染色体SNP位点分布均符合Hardy-Weinberg平衡,不存在连锁不平衡现象。各位点平均杂合度(Ho)为0.423,平均个体识别力(DP)为0.560,平均多态信息含量(PIC)为0.329。90个SNP体系的累积个体识别率(CDP)为(1-1.20×10~(-33)),大于20A体系;三联体累积非父排除率(CPE_(tri))为0.999 999 911,二联体累积非父排除率(CPE_(duo))为0.999 882,均小于20A。结论 90个常染色体SNP检测体系可独立应用于法医个体识别和三联体亲子鉴定,并辅助进行二联体亲子鉴定。     

AutoMate Express™ forensic DNA extraction system for the extraction of genomic DNA from biological samples

Abstract: The AutoMate Express? Forensic DNA Extraction System was developed for automatic isolation of DNA from a variety of forensic biological samples. The performance of the system was investigated using a wide range of biological samples. Depending on the sample type, either PrepFiler? lysis buffer or PrepFiler BTA? lysis buffer was used to lyse the samples. After lysis and removal of the substrate using LySep? column, the lysate in the sample tubes were loaded onto AutoMate Express? instrument and DNA was extracted using one of the two instrument extraction protocols. Our study showed that DNA was recovered from as little as 0.025 μL of blood. DNA extracted from casework‐type samples was free of detectable PCR inhibitors and the short tandem repeat profiles were complete, conclusive, and devoid of any PCR artifacts. The system also showed consistent performance from day‐to‐day operation.     

Comparison of DNA yield after long-term storage of Second World War bone samples

Sample storage is of paramount importance in forensic genetics laboratories since only optimal storage enables successful recovery of DNA from old bones that contain very low amount of severely degraded DNA. When identification of missing persons from skeletal remains is completed, bone sample is routinely stored at -20 °C for long-term storage for retesting in future, if necessary. After molecular genetic analyses of Slovenian Second World War (WWII) victims, small fragments of femurs were stored at -20 °C. Reduction in DNA recovery has been observed in frozen liquid DNA extracts by some authors and the goal of our study was to explore how freezing of bone samples affects the preservation of DNA. To achieve this goal, the difference in DNA yield in extracts obtained from WWII bones analyzed in 2009 (data from published paper) and DNA yield in extracts obtained from the same bones (piece sampled next to the one used in 2009) taken out of the freezer after long-term storage on -20 °C for 10 years was examined, using the same extraction method and the same quantification kit. Up to 100 ng DNA/g of bone powder was obtained from 57 WWII femurs and up to 31 ng DNA/g of bone powder from the same femurs investigated after long-term storage in this study. 0,5 g of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 device (Qiagen) and DNA quantity determined with the Human Quantifiler kit (TFS). Statistical analysis showed significant difference in DNA yield in extracts obtained from WWII bones in 2009 and extracts obtained from the same bones stored at -20 °C after 10 years. As reported for frozen liquid DNA extracts, reduction in recovery of DNA was confirmed for frozen bone samples as well.     

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。     

A Comparison of DNA Extraction Using AutoMate Express™ and EZ1 Advanced XL from Liquid Blood,Bloodstains, and Semen Stains

In this study, DNA was extracted using an AutoMate Express? and an EZ1 Advanced XL from liquid blood, fresh and aged bloodstains, and fresh and aged semen stains. Extracted DNA was quantified by real‐time PCR using the D17Z1 locus. Short tandem repeat typing was performed using an AmpF?STR^® Identifiler kit. The yields of DNA obtained by the AutoMate Express? were higher from fresh bloodstains and fresh semen stains, almost the same from aged bloodstains and aged semen stains, but slightly lower from liquid blood compared with those obtained by the EZ1 Advanced XL. The addition of dithiothreitol or the use of PrepFiler? lysis buffer improved the EZ1 Advanced XL results from fresh bloodstains, but not for liquid blood and aged bloodstains. Our results demonstrated that the PrepFiler? lysis buffer is the main contributor to the higher DNA yields of the AutoM ate Express? for fresh bloodstains.