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。     

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。     

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。     

Determination of DNA yield rates in six different skeletal elements in ancient bones

Current sampling strategy for laboratories typing bones for human identification include samples obtained from femur, tooth and temporal bone. Latest studies suggest that the small bones of the hands and feet were very similar or even better in DNA yield. These bones can be easily sampled with a disposable scalpel and thus reduce potential DNA contamination. The aim of our study was to determine the suitability of metatarsals, metacarpals and phalanges for genetic identification. 48 bone samples from 8 different skeletons (six from 18^th century and two from 3rd century) were obtained from 5 archaeological sites in Slovenia. In each skeleton, 6 different skeletal elements were sampled (temporal bone, molar, femur, metacarpal bone, metatarsal bone and proximal phalanx of the hand), and strict precautions followed to prevent contamination. Half of gram of bone powder was decalcified using full demineralization extraction method. The DNA was purified in a Biorobot EZ1 (Qiagen), DNA content determined with the PowerQuant kit (Promega), and autosomal STR typing performed with the Investigator ESSplex Plus kit (Qiagen). Up to 8.75 ng DNA/g of powder was obtained from samples analyzed. The highest yields were detected in temporal bone and the lowest in femur. The success rate of STR typing was evaluated according to the number of successfully typed loci and a strong correlation between the success rate of STR typing and the amount of extracted DNA was confirmed. For all eight skeletons full consensus genetic profiles were determined from skeletal elements analyzed. Our findings suggest it would be suitable to include metatarsal and metacarpal bones in sampling strategy for human identification although further research is needed to substantiate the findings of this study.     

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。     

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。     

Highly efficient and automated extraction of DNA from human remains using a modified EZ1 protocol

Bones and teeth often represent the only sources of DNA available for identifying human remains. DNA in bones and teeth is generally better preserved than that in soft tissues because of the presence of hard connective tissue with a high level of calcium. Because of the extensive mineralisation, the choice of an efficient DNA extraction procedure is important to minimise the sampling of a high level of minerals and to remove polymerase chain reaction (PCR) inhibitors. Some protocols are available for DNA extraction from bones and teeth as part of the Qiagen EZ1 DNA Investigator Kit using the EZ1 Advanced XL automated purification platform. To improve the efficiency of DNA extraction from skeletal remains, the present study focuses on a modification to these already available protocols. In this study, different bones and teeth collected between 1 and 50 years after death were subjected to DNA extraction using the standard EZ1 protocol, a supplementary protocol, and a modified protocol. The modified approach included a decalcification step, whereas the Qiagen protocols worked directly on non-decalcified powder. In all three procedures, 150 mg samples were used for DNA extraction. We evaluated the quantity of DNA recovered from samples, the presence of any PCR inhibitors co-extracted, the level of DNA degradation, the quality of short tandem repeat (STR) profiles, and the reproducibility of the modified procedure. When compared with the other protocols, the modified protocol resulted in the best recovery of DNA that was free of PCR inhibitors. Additionally, the STR profiles were reliable and of high quality. In our opinion, the decalcification step increases DNA recovery by softening tissues, which allows lysis solutions to act more effectively. Furthermore, the use of two lysis solutions and the variation added to the EZ1 purification step allow for DNA recovery with quality and quantity superior to those of the previously available Qiagen-based protocols. These findings may be helpful solutions to the problems commonly encountered when dealing with difficult samples, such as bones and teeth.

Key points

• Bones and teeth often represent the only sources of DNA for identifying human remains.
• The choice of an efficient DNA extraction procedure is important for maximizing DNA recovery and removing PCR inhibitors.
• This study focuses on modifications to the previously available Qiagen-based protocols.
• The modified protocol enabled the best recovery of DNA, and both quality and quantity were superior to those of the previously available Qiagen-based protocols.
• The STR profiles obtained from samples extracted using the modified protocol were reliable and of high quality.

     

微量口腔脱落细胞检材的DNA检验

目的寻求提高微量口腔脱落细胞检材的DNA检验成功率的简便有效的提取方法。方法对不同载体上的100份微量口腔脱落细胞检材采用小体积Chelex-100法提取DNA,在ABI7500型荧光定量PCR仪上进行定量,同时用IdentifilerTM复合扩增系统扩增,在ABI3130遗传分析仪上进行STR分型。结果从25根饮料吸管上提取的DNA量在0.72～116.7.8ng,16个水杯杯缘提取的DNA量在2.15-142.5ng,31个饮料瓶(罐)口提取的DNA量在1～34.65ng,10根筷子上提取的DNA量在3.35～26.6ng,12个果核中提取的DNA量在0.294～21.4ng,6份吃剩的骨头中提取的DNA量在0.88～5.88ng。100份检材性别及9个以上STR位点分型成功率平均为59.38%。除了使用者的个人原因外,检材的提取送检方式、检材的质地、饮料的性质对提取的DNA量有显著影响,是否加蛋白酶K对提取的DNA量无显著影响。结论采用小体积Chelex-100法可对60%左右的微量口腔脱落细胞检材提取DNA进行STR分型。     

Efficiency evaluation of a DNA extraction and purification protocol on archival formalin-fixed and paraffin-embedded tissue

Formalin-fixed and paraffin-embedded tissue (FF-PET) is an invaluable resource for retrospective molecular genetic studies, but the extraction of high-quality genomic DNA from FF-PET is still a problematic issue. Despite the range of DNA extraction methods currently in use, the association of phenol–chloroform extraction and silica-based purification protocols, reported in ancient DNA studies on archaeological bones, has, to our knowledge, not been used for DNA extraction from FF-PET yet. The present study compared the efficiency of three DNA extraction and purification protocols from two different FF-PET substrates, heart and liver, by using quantitative PCR and multiplex amplification.We showed that the method, using phenol–chloroform and the QIAamp DNA mini^® Kit (Qiagen), was the most effective DNA extraction and purification method and that the DNA quantity extracted from liver is statistically more important than that extracted from heart. Autosomal STR typing by multiplex amplifications gave partial allelic profiles with only small size products (less than 300 bases) amplified, suggesting that DNA extracted from FF-PET was degraded.In conclusion, the protocol presented here, previously described in studies on ancient bones, should find application in different molecular studies involving FF-PET material.     

Full STR profile of a 67-year-old bone found in a fresh water lake

DNA extraction from and DNA typing of fresh water-exposed aged bone specimens poses a challenging task and is not very well examined. This study presents a new method to extract typable DNA from such problematic bone specimens. The procedure comprises low-heat drilling and cryogrinding, mild lysis conditions, and silica-column-based DNA cleaning. DNA quantity is assessed by quantitative PCR prior to short tandem repeat (STR) amplification. The procedure was employed with a 67-year-old tibia bone fragment recovered from a fresh water lake and succeeded to produce a full STR profile using the MPX-SP1 and MPX-SP2 mini-STR kits and a partial profile with 12 successfully amplified STRs using the Identifiler STR kit. The new method for the extraction of DNA from aged fresh water-exposed bone specimens presented herein was successfully applied to prepare DNA of sufficient quality and quantity to generate a full STR profile.     

陈旧性骨骼DNA提取技术的研究

对陈旧性骨骼建立一个高回收率并能除去 PCR 反应抑制物的提取 DNA 的方法。采用 CTAB 法提取 DNA。结果显示,该方法不但能有效去除 PCR 抑制物,而且对水泡、火烧、土埋以及10年左右的骨骼所提取的 DNA 均能成功地进行荧光标记 STR 多基因座扩增检验。实验表明,该方法稳定,重复性好,适合陈旧骨骼标本的 DNA 提取。     

The Successful Recovery of Low Copy Number and Degraded DNA from Bones Exposed to Seawater Suitable for Generating a DNA STR Profile

A universal method allowing for DNA profiling from bones exposed to seawater has not been reported yet. This study refers on the identification of a body immersed in seawater for 8 months. The biological material for identification was the mandibular body, usually characterized by low success rates of DNA analysis. Initially, two extraction protocols were performed with negative results: one used for bones immersed in fresh water and a silica‐column procedure. A third protocol was performed, which combined the extraction of a higher amount of bone powder, the use of multi‐silica‐based extraction columns followed by a concentration step. This protocol allowed to obtain low copy number DNA and to generate a 12‐loci STR profile by combining conventional STR typing and mini‐STR technologies. This protocol could be suitable when human bones have been exposed to severe environmental conditions, and the available nuclear DNA is highly degraded and in low copy number.     

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。     

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。     

自动荧光分析法检验陈旧骨骼和牙齿的vWA和LPL位点

为鉴定陈旧骨骼和牙齿的尸源 ,对DNA的提取方法进行了研究,并建立了vWA和LPL位点的自动荧光分析技术,该法简便、快速、有效、可对存放2、3、4、7年的陈旧骨骼和牙齿成功地进行检验,在检验中能直接确定样品的等位基因     

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.     

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。     

DNA IQ磁珠法结合Maxwell~（TM） 16自动仪提取接触DNA

目的研究DNA IQ磁珠法结合MaxwellTM 16自动仪对接触DNA提取的应用价值。方法 151份案件接触DNA检材95℃裂解后,采用DNA IQ磁珠法结合MaxwellTM 16自动仪提取DNA,然后进行DNA定量和STR分型检测,统计各种类型的接触DNA含量I、PC CT值和STR分型成功率。结果 151份案件接触DNA检材中,除果核平均DNA获得量为9.51ng以外,其它接触检材的平均DNA获得量均大于10ng,烟蒂检验成功率最高为93%,果核检验成功率较低,为60%。所有DNA样品的IPC CT值均在27左右,纯度高。结论大部分接触DNA检材采用DNA IQ磁珠法结合MaxwellTM 16自动仪可提取到足以进行STR分型的DNA。     

Different skeletal elements as a source of DNA for genetic identification of Second World War victims

To this day process of identification of missing persons from skeletonized human remains with help of forensic genetics proves to be complex and challenging. The success rate of genetic identification in bones strongly depends on a combination of various factors, most importantly environmental factors and post-mortem interval. Furthermore, there are individual-specific factors that affect DNA preservation, such as race, gender, age and type of skeletal elements. The goal of our study was to optimize sampling process through determining which skeletal elements are superior in their preservation of DNA in 70-yearold skeletons belonging to victims of Second World War. We sampled different types of bones and teeth from three such skeletons found in Slovenian hidden mass grave Huda jama, 56 elements from each respective skeleton, together 168 elements. With the help of parameters, such as quantity of DNA, degradation rate and typing success, we tried to find the best types of elements to identify the victims. Prior to powdering bones and teeth, we removed contaminants. We decalcified 0.5 g bone and tooth powder followed by extraction and purification of DNA using Biorobot EZ1 (Qiagen). Quantification of obtained nuclear DNA was carried out using PowerQuant kit (Promega) and autosomal STR typing using ESSplex SE QS kit (Qiagen). Best parameters to assess skeletal elements that are superior in their DNA preservation were quantity of DNA and number of successfully typed STR loci. Metacarpal and metatarsal bones proved to be the best, followed by intermediate cuneiform, first distal foot phalanx, talus, petrous bone and tibia. We also created elimination database for persons involved in exhumation, anthropological and genetic analyses and exclude potential contamination.     

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.