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.     

收集提取“物证”样本规范化研究——以物证鉴定样本为切入点

物证是证据种类之一,而物证鉴定样本是在物证鉴定过程中,为了实现物证的种属认定与同一认定而依法提取和保存的、供比对的已知证据材料。物证鉴定样本因鉴定而成为证据,是鉴定意见的重要组成部分。物证鉴定样本在诉讼中具有重要作用。当前,样本收集、提取存有许多不足,需采取行之有效的收集提取方法,以助于物证鉴定制度在内的整个证据制度的完善。     

大白口蘑子实体的化学成分研究

目的 研究大白口蘑(Tricholoma giganteum Massee)子实体的化学成分。方法 大白口蘑子实体的乙酸乙酯部位经过硅胶、RP-18、Sephadex LH-20等多种材料进行分离纯化, 通过现代波谱技术进行结构鉴定。结果 分离鉴定了12个化合物,它们分别为：亚油酸甲酯（1）、亚油酸（2）、麦角甾-4, 6, 8 (14), 22-四烯-3-酮（3）、麦角甾-5, 7, 22-三烯-3β-醇（4）、过氧麦角甾醇（5）、麦角甾-7, 22-二烯-3β, 5α, 9α-三羟基-6酮（6）、麦角甾-7, 22-二烯-3β, 5α-二羟基-6酮（7）、麦角甾-7, 22-二烯-3β, 5α, 6α, 9α-四醇（8）、麦角甾-7, 22-二烯-3β, 5α, 6β, 9α-四醇（9）、麦角甾-7, 22-二烯-3β, 5α, 6β-三醇（10）、3β-O-glucopyranosyl-5α, 6β-dihydroxy-ergosta-7,22-diene（11）、脑苷脂D（12）。结论 化合物1-3、6-12均为首次从该种真菌中分离得到。     

SPE-GC/MS 法分析鱼塘水中的5种农药

目的：建立鱼类中毒案件中鱼塘水5种农药的固相萃取（solid phase extraction,SPE）结合气相色谱－质谱联用仪（GC/MS）的检测方法。方法比较 Oasis HLB cartridge、Bond Elut C18和 SampliQ C183种固相萃取柱提取、净化鱼塘水中5种农药的提取回收率,并考察洗脱剂种类、洗脱剂用量对提取回收率的影响。结果利用 Bond Elut C18固相萃取柱,以3mL 苯作为洗脱剂,鱼塘水中5种农药质量浓度在1~50μg/mL范围内有良好的线性关系,相关系数为0．9962~0．9996,测得的检出限为3．4~26μg/L,提取回收率为61．49%~102．48%,相对标准偏差均≤3．01%。结论 SPE－GC/MS 法灵敏度高、准确度和精密度好,简单快速,溶剂消耗量少,适用于鱼塘水中5种农药的检测。     

气相色谱-串联质谱法分析尿和血中除草剂百草枯

目的建立尿和血中百草枯的离子交换固相萃取-气相色谱-串联质谱分析方法。方法尿样加内标乙基百草枯,用732阳离子交换树脂提取;血样加内标乙基百草枯,用三氯乙酸凝聚蛋白质后取上清液用732阳离子交换树脂提取。提取物用硼氢化钠在水溶液中碱性条件下还原,还原物用有机溶剂提取进行气相色谱-串联质谱法分析。结果尿和血中百草枯的提取率分别为76％和74％,检测限分别为2ng／mL和10ng／mL,尿添加百草枯100ng／mL和血添加百草枯500ng／mL水平的回收率分别为99．6±5．6％和99．3±7．6％（Mean±CV）。结论本文建立的分析方法灵敏度高,能够满足中毒致死案件检验及临床毒物检验的需要。     

法医物证DNA自动化检验技术体系的研究

目的建立自动化工作站同步提取不同种类涉案法医生物检材DNA的新方法。方法选用TECAN Freedom EVO100．4、75—2型自动化提取、加样工作站,采用磁珠法及Chelex-100法对各类涉案生物检材进行DNA提取、PCR扩增、毛细管电泳检测其STR分型,进行比较测试。在“全国公安机关DNA数据库应用系统”中建立并应用实验室信息管理系统（LIMS）模拟实施规范化DNA检案。结果1552份各类检材,采用工作站-磁珠法提取DNA效果最佳,STR检测成功率为95％,工作站-Chelex法为88％;二者分别与其手工提取法比较,成功率无明显差异。92个样本同期检测,自动化工作站较手工操作DNA检案时间可缩减1．25倍。结论工作站域珠法提取涉案检材DNA,可获得满意的STR分型结果。应用LIMS管控,可有效防控污染,明显提高检案效率及鉴定质量。     

3种方法联合运用提取人体脱落上皮细胞DNA

目的寻求提高法医物证检验中附有人体脱落细胞载体检材的DNA检出率的方法。方法根据案件检材的具体条件．选择3种提取方法即Chelex-100法、Chelex-100联合有机法和Chelex-100联合磁珠法。结果采用了分步提取DNA法,可充分、有效地提取微量DNA,提高了人体脱落上皮细胞有效DNA的检出率。结论根据第一步提取方法的检测结果,来调整下一步的提取策略,如是否加以纯化浓缩,可增加人体脱落上皮细胞有效DNA的检出率。     

Extraction of high quality DNA from biological materials and calcified tissues

Calcified tissues, such as bone and tooth, and some other sample types, such as those containing adhesive, present a challenge to standard extraction protocols. We have developed a lysis reagent, BTA™ lysis buffer, which is designed for use with PrepFiler™ Kit reagents. The BTA™ lysis buffer disrupts calcified tissue matrices and achieves effective extraction of DNA from pulverized bone and tooth samples. In addition, the BTA™ lysis buffer mildly but efficiently extracts DNA from challenging substrates like tape, chewing gum, and cigarette butts and, as with bone and tooth, DNA from these lysates is purified using established PrepFiler™ reagent extraction protocols.We successfully extracted DNA from powdered human bone samples, chewed gum and smoked cigarettes using BTA™ lysis buffer. Extraction yields for bone, gum and cigarette samples tested were consistent and reproducible. This extraction method efficiently removed potential PCR inhibitors from all samples tested, and C_T values for the internal PCR control of Quantifiler^® Human DNA Quantification Kit were consistent and within the normal range. The DNA extracted from these samples also provided conclusive profiles that were free of PCR artifacts when amplified using the AmpF?STR^® Identifiler^® PCR Amplification Kit. The protocol is easily adapted for automation.     

Case report of a homicide resolved 15 years later: The robustness of Chelex extraction

DNA typing techniques is one of the most advanced tools for human identification. During the last 10 years, a great number of methods for DNA extraction and analysis have been introduced to forensic genetic, with considerable success but also with considerable controversy. The success and validation of a criminal investigation are very closely related to the process used for obtaining and preserving biological evidence.We report the strategy that we employed to analyze evidences belonging to a homicide happened in Brescia (Italy) in 1992, not resolved at that time, with the forensic genetic analysis. After 16 years the analysis were conducted on DNA samples extracted with Chelex maintained at −80 °C, bloodstain, and biological specimens of perpetrators. Standard autosomal and Y-chromosome STR analysis identified the persons involved and victim's profiles. This case is of interest as a demonstration of a more successful application of DNA typing in well conserved DNA samples than in bloodstains kept in the Court Office.     

Successful STR and SNP typing of FTA Card samples with low amounts of DNA after DNA extraction using a Qiagen BioRobot EZ1 Workstation

FTA Cards (GE Healthcare) have been used for more than 4 years in Denmark for the collection of buccal cells as reference samples in crime cases. Semi-automated protocols for STR typing of DNA on punches of FTA Cards are routinely used. In average, full STR profiles were generated from approximately 95% of the FTA Cards with a standard punching protocol, while partial or no STR profile were obtained from 5% of the samples. Here, the Qiagen BioRobot^® EZ1 Workstation (Qiagen) and the EZ1 DNA Investigator Kit (Qiagen) was used to extract DNA from 29 FTA Cards from which a complete STR profile was not generated with the standard punching protocol. All 29 samples were successfully typed with the AmpF?STR^® Identifiler™ PCR Amplification Kit (Applied Biosystems) and with the SNPforID 49plex SNP assay. The lowest amount of DNA that resulted in complete STR and SNP profiles was 80 pg. The STR and SNP profiles were identical to those generated from another sample collected from each of the 29 individuals.