微量体表脱落上皮细胞的DNA检验

目的建立微量体表脱落上皮细胞的DNA检验方法. 方法采用Chelex-100法提取DNA,以Microcon-100纯化柱纯化浓缩DNA,用Profiler Plus 试剂盒PCR扩增后用310基因分析仪检测. 结果 10种常见粘附有体表脱落上皮细胞的样本100个,其中7种70个样本成功检测到10个STR位点的分型,检出率为100%,其余3 种样本检出率分别为50%、20%、10%,将该方法应用于2例实际检案,取得满意效果. 结论所建立方法稳定可靠,易于操作,适用于多种检材,为微量体表脱落上皮细胞的DNA检验提供了确实可行的检验方法.     

带血手套腕部微量上皮细胞DNA检验1例

PCR技术应用到生物物证的检验,尤其是应用荧光复合STR基因座进行分析,使微量生物检材的检测成为可能[1]。在刑事案件中,与人体表接触过的衣服、手套等常粘附有体表脱落上皮细胞,如能对此进行检验并成功获得DNA分型数据,将为案件的侦破提供重要的线索和证据。通常情况下,因脱落上皮细胞量非常少,在检验过程中又缺乏稳定的方法,常常导致检验结果不理想。本文作者通过对一例棉线手套腕部上的脱落上皮细胞成功进行STR分型,为极微量体表脱落上皮细胞的检验提供了一种切实可行的方法。1案例2005年12月,某商店内发生入室抢劫案,两名妇女被杀害…     

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。     

PCR产物纯化增强STR分型强度的研究

目的研究PCR产物纯化对微量DNA的STR分型的影响。方法使用25pg/μL的9947标准基因组DNA为模板,标准程序扩增和STR分型检测。设立对照组A和实验组B、C、D。实验组分别使用分子筛(Qiagen DyeEX2.0spin kit)、超滤膜(Amicon Ultra-0.5,100KD)、亲和层析(Qiagen MinElute Column)3种DNA纯化方法,对照组不做任何处理。结果与对照组相比,3种方法均可显著提高STR分型强度(P峰高<0.001),平均峰高约为对照组的4倍,并且3种方法对提高STR分型强度无显著差异(P峰高=0.249)。结论 PCR产物纯化能显著提高微量DNA的STR分型强度,可用于骨骼、脱落细胞等微量DNA检材的检验。     

两种DNA提取方法检测微量陈旧汗斑1例

与人体接触过的衣、物常常附有体表脱落的上皮细胞成分。有试验证明,人体只要接触物体5秒钟以上,即有可能细胞遗留在物体上,也就是说,有可能留下核DNA及线粒体DNA[1]。在刑事案件中,如能成功地对有关物品上粘附的脱落上皮细胞的DNA检验,便可为案件的侦查和审判提供重要的线索和证据[2]。作者在实际检案中遇到一例微量陈旧汗斑,通过联合应用两种DNA的提取方法并进行了对比,解决了该案的难题。报道如下:1简要案情2003年初,某女被杀害。现场提取男士夹克衫一件,领后部有可疑油渍样斑迹,考虑此处有穿衣者留下的汗斑。2检验2.1检验材料犯罪…     

DNA-STR分型检验人体脱落上皮细胞2例

作者结合 2个典型案例 ,对一些特殊载体上的脱落上皮细胞成功地进行了STR检验分型 ,并就检验中的相关问题进行讨论 ,旨在进一步提高办案人员对此类检材应用价值的注意。1　案例资料案例 1　 2 0 0 3年 1月在某旅社内 ,犯罪嫌疑人王某将徐某 (女 ,2 7岁 )掐颈致死后 ,用打火机烧灼电视机电线并用手拉成多节 ,用于捆绑死者。用纱线两步擦试法转移电视机电线上上皮细胞 ,用Chelex 10 0法提取上皮细胞DNA ,Microcon 10 0纯化柱处理 ,用AmpFISTR profilerPlusTM 系统复合扩增试剂盒进行STR位点的复合扩增 ,扩增产物经ABI 310型基因分析…     

三种磁珠法提取脱落细胞DNA的比较

正法医DNA鉴定中的脱落细胞检材是指含有人体皮肤黏膜脱落上皮细胞的检材,是当前刑事案件现场最有可能提取到的生物物证。目前关于脱落细胞提取纯化方法对比的报道~([1-3])较多,但相同提取方法效果横向比较的研究较少。本研究对IQ磁珠、M48磁珠、Prep Filer磁珠三种常见的磁珠法提取脱落细胞DNA的检验效果进行比较,为法庭科学实践提供参考依据,现报道如下。     

脱落上皮细胞类生物检材的自动化提取

目的建立一种自动化提取脱落上皮细胞类生物检材DNA的方法。方法附着于不同载体上的脱落上皮细胞类生物检材共278份,应用Eppendorf epMotion 5075LH工作站结合DNA IQTM系统提取模板DNA,并用Identifiler试剂盒进行STR检验。结果在278份被检的生物检材,其中126份检材获得13个基因座以上的STR分型,不同类型的检材其检出率不相同,最高达73.44%,最低为10.89%。结论脱落上皮细胞类检材应用自动化工作站提取DNA模板可在法医日常检案中广泛应用。     

一种收集衣服上脱落细胞的新方法

目的建立一种生物脱落细胞的微量提取新方法。方法利用一套自制的“生物细胞提取仪”无损提取衣物等载体上的人体脱落细胞,采用Chelex-100法提取DNA,用不同的试剂盒进行STR复合扩增检验。结果10例检材都得到16个基因座成功分型。结论用该方法提取微量细胞DNA,可获得满意的DNA分型。     

人体脱落上皮细胞的荧光标记STR分型

荧光标记STR分型技术在法医物证检验中已经得到广泛应用 ,但在实际工作中 ,含有微量人体脱落上皮细胞的特殊载体在很多情况下被忽略 ,关于皮肤脱落上皮细胞的DNA检验比较少。本文作者结合案例 ,对一些特殊载体上的脱落上皮细胞成功地进行了STR检验分型 ,并就检验中的相关问题进行讨论 ,旨在进一步提高办案人员对此类检材应用价值的注意。案例资料【案例 1】　 2 0 0 3年 1月在某旅社内 ,犯罪嫌疑人王某将徐某(女 ,2 7岁 )掐颈致死 ,后用打火机烧灼电视机电线再用手拉成多节 ,欲用于捆绑。鉴定人员在现场采用纱线两步擦拭法转移电视机电…     

Male DNA typing from 25-year-old vaginal swabs using Y chromosomal STR polymorphisms in a retrial request case.

We report here the application of Y chromosomal DNA analysis in a retrial request case, raised officially by Sapporo High Court, Japan, of a condemned criminal whose capital punishment has been suspended. DNA was extracted from mixed seminal/vaginal secretion stains collected 25 years ago from two raped and murdered victims, and Y chromosome STR loci (DYS19, 390, 393, YCAII) were amplified and sequenced to clarify the DNA type of the rapist. Alkaline proteinase and sodium hydroxide were used before phenol/chloroform extraction to achieve high quality DNA from very old samples. In addition, amplified fragments of DYS19, DYS390, and DYS393 were sequenced using an automated DNA sequencer. Four Y STR DNA types detected from vaginal swabs were found identical to those of the accused criminal and confirmed that the two rape and murder cases had been committed by the same person. Sapporo High Court accepted the results and rejected the retrial request in February 1998.     

DNase-I纯化结合碱性裂解法提取混合斑精子DNA

目的研究脱氧核糖核酸酶I(DNase-I)纯化结合碱性裂解法提取混合斑精子DNA的方法在法医学中的应用。方法收集79份性犯罪案件混合斑检材,分别用DNase-I纯化结合碱性裂解法和差异裂解法提取精子DNA,采用STR荧光标记复合扩增体系进行16个STR基因座分型,并比较检验结果。结果应用DNase-I纯化结合碱性裂解法提取精子DNA,64例检材分型成功;应用差异裂解法提取精子DNA,57例检材分型成功;两种方法比较结果存在显著性差异(P=0.039),DNase-1纯化结合碱性裂解法提取精子DNA的STR分型成功率更高,成本低廉。结论DNase-I纯化结合碱性裂解法提取混合斑精子DNA可提高检验成功率,操作简便,快速,易于自动化,适于法医学个体识别鉴定。     

DNase-Ⅰ纯化结合碱性裂解法提取混合斑精子DNA

目的研究脱氧核糖核酸酶I(DNase-I)纯化结合碱性裂解法提取混合斑精子DNA的方法在法医学中的应用。方法收集79份性犯罪案件混合斑检材,分别用DNase-I纯化结合碱性裂解法和差异裂解法提取精子DNA,采用STR荧光标记复合扩增体系进行16个STR基因座分型,并比较检验结果。结果应用DNase-I纯化结合碱性裂解法提取精子DNA,64例检材分型成功;应用差异裂解法提取精子DNA,57例检材分型成功;两种方法比较结果存在显著性差异(P=0.039),DNase-1纯化结合碱性裂解法提取精子DNA的STR分型成功率更高,成本低廉。结论DNase-I纯化结合碱性裂解法提取混合斑精子DNA可提高检验成功率,操作简便,快速,易于自动化,适于法医学个体识别鉴定。     

Recovery of genomic DNA from archived PCR product mixes for subsequent multiplex amplification and typing of additional loci: forensic significance for older unsolved criminal cases

A method for genomic DNA recovery from different types of PCR product mixes suitable for multiplex amplification and typing using the Profiler Plus STR typing system has been investigated. The application of this method is of significance in cases where the original DNA samples have been exhausted due to repeated typing analyses in an effort to maximize their evidentiary value. Such cases typically involve samples analyzed using the available DNA typing systems of the time which gave a markedly lower power of discrimination, either alone or in combination, compared to that of modern multiplex STR typing systems. It was found that an effective method for recovering genomic DNA from HLA-DQA1 +PM and CTT triplex amplification mixes, suitable for reproducible achievement of the complete Profiler Plus profile, involved the use of Amicon Microcon-100 microconcentrators. Interestingly, this method was not required to achieve the complete nine STR profile using D1S80 amplification mixes.     

An analysis of data curated from 5 years of identifying human remains

An archive of 5 years of cases involving the identification of human remains was curated, collecting information on: The sample type submitted, the number of STR loci yielding interpretable results, the kinship challenge posed, and the outcome for the case. A total of 129 cases of remains ID were investigated using manual DNA extraction and recovery methods with amplification of STR markers using the Power Plex 21 multiplex STR kit from Promega Corp. In 52 cases, blood spots collected by the ME were provided as sample and in 100% of those cases, probabilities of relatedness to the reference samples was ≥99%. In 77 cases, tissue other than blood was provided as a source of DNA. These other samples were grouped categorically into long bones (femur and tibia; 40 cases), skull bones/teeth (11 cases), other bones (16 cases), and tissue (normally adherent to bone) (10 cases). Reference samples provided for cases included alleged parents or child(ren) of the victim (86 cases), alleged full siblings of the victim (38 cases), or alleged second-order relatives (five cases). The overall success rate in confirming the identity of the source of the remains in these cases was 89.2%. Our results demonstrate that a laboratory can be often successful identifying human remains using methods easily implemented in any DNA typing laboratory.     

An Optimized DNA Analysis Workflow for the Sampling,Extraction, and Concentration of DNA obtained from Archived Latent Fingerprints

DNA profiles have been obtained from fingerprints, but there is limited knowledge regarding DNA analysis from archived latent fingerprints—touch DNA “sandwiched” between adhesive and paper. Thus, this study sought to comparatively analyze a variety of collection and analytical methods in an effort to seek an optimized workflow for this specific sample type. Untreated and treated archived latent fingerprints were utilized to compare different biological sampling techniques, swab diluents, DNA extraction systems, DNA concentration practices, and post‐amplification purification methods. Archived latent fingerprints disassembled and sampled via direct cutting, followed by DNA extracted using the QIAamp® DNA Investigator Kit, and concentration with Centri‐Sep? columns increased the odds of obtaining an STR profile. Using the recommended DNA workflow, 9 of the 10 samples provided STR profiles, which included 7–100% of the expected STR alleles and two full profiles. Thus, with carefully selected procedures, archived latent fingerprints can be a viable DNA source for criminal investigations including cold/postconviction cases.     

汗潜指印的STR分型检测在案件中的应用

目的探索案例中所涉及的汗潜指印DNA的提取和检验方法。方法采用Chlex-100法提取DNA,进行STR复合扩增,通过毛细管电泳检测荧光信号。结果案例中涉及的汗潜指印在ProfilerPlus试剂盒10个基因座的分型检测均获成功。结论含有汗潜指印的检材的发现和正确提取对最终检测成功至关重要。     

激光显微捕获技术用于尿液脱落细胞DNA检测

目的采用激光显微捕获技术（LCM）捕获尿液脱落细胞,并进行STR分型。方法收集10份健康成人尿液样本,根据储存时间分组,其中新鲜尿液组（≤24h）分别采用Chelex-100及LCM联合DNA IQTM提取法提取DNA,储存尿液组（〉24h）再分为4℃组和室温组,分别在4～30d内不同时间点采用LCM联合DNA IQTM提取法提取DNA;各组提取的模板DNA进行扩增及SRT分型检验。结果新鲜尿液组采用LCM联合DNA IQTM提取法提取DNA,所有样本均可检出全部基因座（16个）,采用Chelex-100法则在部分基因座上出现等位基因丢失、非特异性扩增、峰值低等现象;4℃储存10d和室温储存4d以内的尿液经检验可明确判读12个以上基因座,4℃20～30d及室温7d,可检出7个以上基因座。结论 LCM技术可用于尿液检材的DNA分型检验,且检材应尽可能4℃保存并尽快检验。     

强奸致孕形成部分性葡萄胎的检验分析

目的探讨强奸致孕案葡萄胎组织的DNA检验和STR结果分析。方法用DNA Typer TM15试剂盒对犯罪嫌疑人、受害人血样和流产胚胎组织进行荧光复合STR基因座扩增检测。结果检测的一例流产胚胎组织DNA的STR峰谱表现出部分3条带,且3条带中有两个等位基因来源于犯罪嫌疑人,另外一个来源于受害人,推断为单卵双精子受精的部分性葡萄胎。结论应用STR分型技术可以推断葡萄胎的DNA来源和受精类型,为亲缘关系鉴定提供依据。     

Direct STR amplification from whole blood and blood- or saliva-spotted FTA without DNA purification

The DNA purification step has been thought to be essential for typing of STR DNA. However, this process is time-consuming, and there is a risk of unexpected cross-contamination during purification. We report a new method for direct short tandem repeat (STR) amplification using a newly developed direct PCR buffer, AnyDirect, which can amplify STR loci from whole blood and blood- or saliva-spotted FTA cards without DNA purification. The autosomal and Y chromosomal STR loci were analyzed for whole blood and blood or saliva spots of random individuals, followed by comparison of the results with those of corresponding purified DNA. The results from whole blood and blood spots showed perfect concordance with those from purified DNA without allele or locus drop-out. However, in the case of saliva spots, no amplification or locus drop-out was observed in some of the samples, which offers a topic for further study. Additionally, some commercial hot-start DNA polymerases other than AmpliTaq Gold DNA polymerase were also found to be compatible with this buffer system. Therefore, this direct PCR buffer was demonstrated to be useful for fast forensic DNA analysis or criminal DNA databases for which there is no need to store DNA samples.