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™.     

DNA Purification Cell Lysis and Wash Step Modifications for Low-Template DNA Sample Processing,

As DNA technology becomes increasingly sensitive, forensic laboratories are receiving more low-template DNA samples. These samples, already low in DNA content, become even more challenging to process as the available DNA becomes further reduced during the extraction step. In this study, two extraction modifications were tested to determine if the cause of DNA loss could be identified and mitigated. A double lysis technique was used to test for DNA loss in the sample collection substrate, and lysate eluates were re-extracted to determine DNA loss from inefficient binding to the silica column. Both modifications showed DNA was lost at these steps. However, resulting STR profiles from these samples had fewer peaks and lower peak heights when compared to samples processed with no extraction modifications. Overall, the potential benefits of adding these extraction modifications for low-template DNA sample processing are not enough to justify the risk associated with additional manipulation.     

法医DNA分析的其他应用

综述了法医DNA的多种用途,如人死后根据体内DNA变化情况推断死后时间间隔,利用线粒体DNA4977bp的缺失以及端粒的长度推断年龄;利用昆虫mtDNA的细胞色素氧化酶亚基I进行昆虫种属鉴定,以帮助确定人的腐尸死亡时间;利用一些食血昆虫体内的人DNA帮助了解与案件的关系;利用DNA标记进行动物如犬、牛、马及猪等个体识别与亲子鉴定,解决因动物引起的纠纷,或者利用DNA分析嫌疑人周围的动物毛发、植物及土壤微生物等,确定嫌疑人与案件的关系。     

Genetic identification of biological samples buried in clay soil during summer and winter seasons

The analysis of a biological samples found in crime scenes can be a challenging task. Minute amount of DNA, degraded DNA and the presence of inhibitors in biological samples, can interfere with the achievement of a complete genetic profile.Chelating resin, silica membranes and silica-coated with paramagnetic resin were the techniques used in this study for the isolation and purification of DNA. Our aim was to find out the best DNA extraction method for application in the presence of biological samples buried in clay soil.     

Validation of the InnoXtract™ DNA extraction method for bone,teeth, and rootless hair

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.     

Visualising DNA transfer: Latent DNA detection using Diamond Dye

A central question is ‘how did DNA get there’? To help answer this, we visually monitored and recorded DNA transfer from one substrate to another. When an individual touches a substrate, traces of their DNA are transferred (primary/direct) which can then subsequently be transferred to a second substrate (secondary/indirect). Currently DNA transfer and how much remains can only be determined by collecting the biological material from the substrate, isolating the DNA and quantifying the amount recovered. However, Diamond™ Dye (DD) enables such DNA transfer events to be visualised by monitoring the movement of cellular material.We examined primary and secondary DNA transfer using aluminium as a primary substrate with cotton, polyester, aluminium and plastic as secondary substrates and four contact types between two substrates (passive, pressure, friction and friction with pressure). Participants pressed their index finger against the aluminium for 15 s and then DD was applied to the area of contact; cellular material was detected via a fluorescence microscope. Contact between that substrate and a second substrate was performed, using one of the four contact types. After this contact between substrates each was viewed microscopically and transfer of cellular material was recorded.Cellular material could be recorded as having transferred from one substrate to another. Substrate and contact type had an effect on the extent DNA transfers. DNA transferred at a high rate with aluminium as a primary substrate and cotton, polyester and aluminium as secondary substrates when pressure with friction was applied. This information expands our understanding of how DNA transfers and which factors affect it, thus assisting greatly with activity level reporting as to how DNA came to be where it was found.     

Nondestructive Methods for Recovery of Biological Material from Human Teeth for DNA Extraction

The extraction of DNA from human skeletal remains applied to forensic, and evolutionary studies do not exclude risks, which are to be evaluated when working with unique specimens that could be damaged or even destroyed. In the present study were evaluated several nondestructive methods for recovering DNA instead of the most currently used pulverization method. Three different procedures to access inside the dental pieces (occlusal perforation, cervical perforation, and cervical cut) have been compared with the aim of recovering as many cell remains as possible to carry out a DNA extraction. Given the DNA quantitation results, a method was proposed that consists of a cervical cut to facilitate the access to the pulp cavity and a subsequent filing of the root canals down to the apex of the dental root. This methodology allows the recovery of both mitochondrial and nuclear DNA, with the minimum deterioration for the dental pieces.     

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.     

DNA芯片技术的发展及其应用

介绍了DNA芯片技术的制备和工作基本原理,以及其在生命科学、医学及法庭科学中的应用.     

人类DNA小卫星区域RNA探针制备及应用的研究(Ⅰ)──pGEM-4Z-MYO亚克隆的构建

利用DNA重组技术，由pUC19－MYO质粒中获得MYO小卫星DNA探针片段，将其插入到具有RNA聚合酶启动子的pGEM－4Z质粒的多克隆位点，从而构建了pGEM－4Z一MYO亚克隆，为制备RNA探针并将其应用于DNA指纹检测，提高DNA指纹的敏感性和小卫星DNA多态性的检出率奠定了基础。     

应用硅珠法提取陈旧骨骼DNA

骨骼相对于人体其他的组织脏器腐败降解慢,是进行高度腐败及白骨化样本DNA检验的唯一检材。但骨骼DNA的提取比较困难,本文运用传统的硅珠法提取骨骼DNA取得了很好的效果。1材料与方法1.1样本1~4年水浸泡、土埋的股骨、肱骨等样本,来自本实验室日常检案检材。1.2方法1.2.1骨骼DNA提取用水洗净骨骼表面,晾干。锯成小块,用锉打磨内外表面。去离子水、乙醇和5%bleach浸泡、晾干,磨成粉末。取骨粉5g;加入0.5mon/L EDTA(pH8.0)溶液,搅拌均匀置于摇床上脱钙12h,去离子水洗两遍,去上清,重复4次[1];沉渣中加入适量裂解液(6mon/L GuSCN,20mo…     

DNA技术侦破一特大系列强奸案

１案件资料近５年在沈阳市于洪区某地连续发生数十起强奸案，案犯体貌特征、作案手段相似，最显著的特征是“小平头”，因此将该系列案件称为“小平头”系列强奸案。该案引起了沈阳市各级公安部门的高度重视。１９９９年８月９日，“小平头”再次作案，将居住在于洪区的李某（女，１８岁）强奸。笔者对该案精斑进行９个STR位点的基因分型并存档，并以此为依据排除曹某等多名嫌疑人。２０００年７月５日，笔者对新发现的嫌疑人王某血样进行DNA鉴定，并与微机案件库中１９９９年８月９日李某被强奸案中精斑谱带比对，其结果见表１，表２．…     

利用烟头DNA鉴定破案1例

1案例简介2002年1月5日凌晨,宜昌市某村发生一起蒙面入室抢劫案,受害人罗某夫妇被一伙蒙面歹徒持刀砍伤,并抢走部分财物。接到报案后,区公安分局侦技人员立即赶赴现场开展侦破工作,经勘察现场,仅在距中心现场60余米处的一田坎下提取到数枚烟头,同时在侦察中发现外来人员向某与李某有作案嫌疑,但李某去向不明,传唤向某,因无确凿证据,向某拒不承认到过现场和参与作案。为揭露和证实犯罪,采取向某血样、李某妻子与女儿血样与现场烟头进行PCR检验。2检验用chelex-100提取现场烟头咬痕处唾液斑DNA和向某血样DNA、李某妻子及女儿血样DNA,经ep…     

DNA检验和家系分析直接侦破命案1例

1简要案情文某(女,6岁半),2006年1月13日下午,在本村玩耍至下午6时家人找其吃饭时发现失踪。2006年2月6日下午,在本村文某某的柴草屋里发现尸体。尸体俯卧于地上,上面压着砖块和稻草,裤子脱至双脚踝关节上方。尸体高度腐败,面部及胸前局部白骨化,臀及大腿局部形成尸蜡。未发现开放性损伤及骨折,内脏组织已溶解,无法判断死因。在死者长裤(未穿内裤)的裤裆处提取有毛囊的阴毛1根。2 DNA检验和分析对提取的阴毛和排查出的22名嫌疑对象血样进行检验。常染色体STR分型取22嫌疑对象血样与现场提取的阴毛(毛囊)进行常染色体STR检验,结果22名嫌…     

郑州地区食尸性苍蝇及幼虫mtDNA中COⅡ基因序列分析

目的 用mtDNA中细胞色素氧化酶辅酶Ⅱ(COⅡ)基因序列鉴定法医学中常见食尸性苍蝇及其幼虫的种类. 方法 收集郑州地区大白鼠尸体上的苍蝇及其幼虫,提取DNA,PCR扩增CO Ⅱ序列,测序,用Clustalx和MEGA 4.0软件对基因序列进行比对分析及构建系统进化树.结果 成虫与幼虫基因差异不明显,CO Ⅱ基因序列可以对棕尾别麻蝇、巨尾阿丽蝇和亮绿蝇进行鉴定,铜绿蝇与丝光绿蝇进化距离较近,CO Ⅱ序列不能将他们区分开,同时还发现巨尾阿丽蝇和亮绿蝇存在种群单核苷酸多态性. 结论 mtDNA中COⅡ序列能有效鉴定郑州地区部分常见食尸性苍蝇的种类,方法 简便、准确.     

改良高盐低pH方法提取陈旧大麻DNA初探

目的探讨建立室温保存10年队上大麻干叶及大麻树脂DNA提取方法。方法采用SDS及改良高盐低pH方法，改变提取缓冲液中β-巯基乙醇终浓度，增加用酚、氯仿快速抽提过程，提取新鲜和陈旧大麻（树脂）DNA，应用大麻叶绿体trnLintron引物进行PCR，琼脂糖凝胶电泳法检测扩增产物。结果用高盐低pH方法获得了10年以上大麻干叶及树脂清晰的电泳图谱，其中成功提取了1份23年陈旧大麻的DNA。结论高盐低pH方法操作简便、实用，可望用于陈旧、微量大麻植株的DNA检测，对于涉毒案件中特殊大麻标本的检验具有一定意义。     

DNA亲子鉴定破案的威力

1案例资料2002年1月27日,某市一粮站家属区姚某(女,49岁)及女儿魏某(14岁)被人杀死在家中。经勘查,提取现场不同地点多处血迹送检,经血痕初步检验,死者姚某及魏某均为“O”型血。死者姚某的丈夫也为“O”型血。在姚某家室内提取的多处不同地点血迹中大多数检验为O型,在客厅地上及卧室桌子上:大立柜内报纸上等几处却检出A型人血。遂寻找与现场有关的犯罪嫌疑人田某(男,21岁)。由于自案发后田某失踪无法与田某血作DNA同一认定。为了确定现场A型人血是否为田某所留,作者采取田某父亲、母亲血做DNA亲子鉴定。检验和结论:选取死者姚某家大立…     

美国“DNA行动计划”对我国的启示

DNA检测技术问世已近30年,在侦查破案和维护司法公正方面发挥着越来越重要的作用。随着DNA检测范围的不断扩大、案件数量的急剧增长,人们发现了许多制约DNA充分发挥证据效力的问题。为了更有效地利用DNA证据,最大限度发挥DNA技术打击犯罪和保护无辜的科技威力,从2004年起,美国开始实施为期5年、耗资10亿美元的“总统DNA行动计划”。这一划时代的DNA国家战略规划成为美国司法部门应用和发展DNA技术一个全面的行动纲领,同时．该项目的实施进展情况也为我国当前和今后更好地应用DNA技术提供了许多宝贵的启示和借鉴。     

从法医相关性昆虫体内提取人类DNA的研究进展

本文从嗜血性昆虫、嗜尸性昆虫、嗜骨性昆虫及实验技术等多角度阐述了从法医相关性昆虫嗉囔中提取人类DNA的可行性及国际研究和应用进展。     

DNA亲缘关系分析在凶杀案件中应用1例

1案例资料2005年9月6日,新疆某县母女俩被杀死在家中。现场勘验时在卧室一长条桌抽屉上发现1枚残缺汗液指纹上粘附微量可疑斑迹,分析极可能为作案人所留,遂将其提取并进行DNA检验,确定为男性个体所留,将其与多名重点嫌疑人的血迹进行STR基因型比对,结果现场指纹上斑迹的STR分型与嫌疑人中阿某(维吾尔族,男,57岁)虽可排除同一,但相似度大大高于无关人群,因此推测作案人有可能与阿某具有亲缘关系。进一步检验住在当地的阿某其他男性亲属(两个兄弟和1个儿子)及其妻子的STR分型,结果均与现场斑迹STR分型不一致,但都提示可能有亲缘关系,进…