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。     

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。     

陈旧骨骼及牙齿的性别检验

为分析陈旧骨骼及牙齿的性别，建立了从陈旧人骨骼和牙齿中提出DNA的方法。通过PCR技术扩增，得到X－Y染色体上的单拷贝同源基因片断（AmelogeninGene）。结果显示，从死亡后2年、4年、7年和10年的4个骨骼和牙齿样品中，均成功地获得了特异的PCR扩增片段。女性为单一的106bp条带，男性为106bp和112bp两条谱带。其快速、简单、可靠，为陈旧骨骼和牙齿的性别鉴定提供了方法。     

陈旧骸骨DNA身份鉴定的法医学进展

骨骼和牙齿是重要的生物样本,由于结构特殊,抗降解能力强,是自然条件下保留DNA理想的生物检材。在历史人物鉴定案例中,陈旧的骨骼和牙齿样本常常是唯一的生物检材,但其DNA呈微量、毁损和不同程度降解状态,需要经过特殊的实验处理以实现身份鉴定。本文综合近年来相关研究报道,从采集检材、DNA提取、DNA富集、检测分析方案等方面进行综述,以期促进陈旧骨骼和牙齿检测体系的进一步发展和完善。     

常染色体STR及线粒体测序用于陈旧性骨骼DNA一例

陈旧性骨骼是法医学实践中一种检验难度比较大的生物检材,常常由于DNA的降解造成检验的失败。本文通过优化陈旧性骨骼的DNA提取条件,对4例埋葬38年的尸骨进行了STR分型和线粒体测序检验．为当事人成功解决了已故亲人尸骨的认定。现报道如下。     

MagAttract(R)M48试剂盒在案例骨骼检验中的应用

陈旧性骨骼DNA检验的关键环节之一是模板DNA的提取及纯化,本文采用MagAttract(R)M48 DNAManual试剂盒(QIAGEN公司,简称M48试剂盒)对陈旧降解骨骼样本进行检验,结果如下.     

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。     

MagAttract~M48试剂盒在案例骨骼检验中的应用

<正>陈旧性骨骼DNA检验的关键环节之一是模板DNA的提取及纯化,本文采用MagAttractM48 DNAManual试剂盒(QIAGEN公司,简称M48试剂盒)对陈旧降解骨骼样本进行检验,结果如下。1材料与方法1.1样本及预处理肱骨、胫骨各1根,取自2具无名白骨化尸体,尸体发现地点分别为树林和自家房内。根据调查分析     

10年陈旧软骨DNA检验1例

陈旧骨骼样本骨组织中DNA含量少且高度降解，DNA提取不仅有一定的难度，而且检验周期相对较长。笔者通过对长达10年的骨骼上附着的软骨成功进行DNA检验，现介绍如下。     

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。     

STR-genotyping from human medieval tooth and bone samples

We extracted the DNA contained in samples of bones and teeth from 10 skeletons excavated from the Gravette site (400-1000 AD, south of France). Ancient DNA was analysed by autosomal short tandem repeats (STRs). The DNA present in these ancient remains appeared very degraded, but nevertheless, better conserved in tooth than in bone samples. Moreover, we showed that the DNA extracted from ancient dental pulp was not exempt from polymerase chain reaction (PCR) inhibitors, which could result from extreme DNA fragmentation. An adapted protocol with a supplementary step of purification removed this inhibition.     

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.     

A Simple and Efficient Method of Extracting DNA from Aged Bones and Teeth

DNA is often difficult to extract from old bones and teeth due to low levels of DNA and high levels of degradation. This study established a simple yet efficient method for extracting DNA from 20 aged bones and teeth (approximately 60 years old). Based on the concentration and STR typing results, the new method of DNA extraction (OM) developed in this study was compared with the PrepFiler? BTA Forensic DNA Extraction Kit (BM). The total amount of DNA extracted using the OM method was not significantly different from that extracted using the commercial kit (p > 0.05). However, the number of STR loci detected was significantly higher in the samples processed using the OM method than using the BM method (p < 0.05). This study aimed to establish a DNA extraction method for aged bones and teeth to improve the detection rate of STR typing and reduce costs compared to the BM technique.     

Nuclear DNA typing from ancient teeth

Because of the adverse effects that diagenesis exert on ancient skeletal remains, DNA from these samples is often compromised to the point where genetic typing can be challenging. Nevertheless, robust and reliable methods are currently available to allow successful genotyping of ancient specimens. Here we report nuclear DNA-based methods and typing strategies used to analyze 2 human skeletons from a medieval burial. Reliable DNA nuclear profiles were obtained from teeth, whereas mitochondrial DNA analyses in bones were inconclusive. A complete nuclear mini short tandem repeat profile was obtained from a well-preserved premolar, but only a partial one from the femur. Increasing the sensitivity of the polymerase chain reaction system allowed a full profile from the latter, but the presence of artifacts reinforced the idea that the interpretation of this kind of analysis must be performed with caution. The results presented here also indicate that DNA from dental pieces can be better preserved than from bones, even in the case of well-preserved long bones with thick cortical tissue such as the femurs, and have a better chance of successful genetic typing, probably because of the high degree of protection conferred to the DNA by the enamel.     

DNA extraction: an anthropologic aspect of bone remains from sixth- to seventh-century ad bone remains

In the archeological site of the early Christian Episcopal complex of Saint Peter, in Canosa di Puglia (Bari, Italy), during the operations of archaeological excavations, tombs were discovered. They were dated between the sixth and seventh centuries ad with carbon 14 methodology. Five skeletons were found in the 5 tombs: 28A: male individual, 43 years old. The height was 170 cm; the biomass was 65.7 kg. The analysis of the bones indicated several noteworthy pathologies, such as a number of hypoplasia lines of the enamel, the presence of Schmorl hernias on the first 2 lumbar vertebrae, and the outcome of subacromial impingement syndrome. 28E was a male individual, with a biologic age of death of between 44 and 60 years. The height was 177 cm. He had a posttraumatic fracture callus of the medial third of the clavicle, with an oblique fracture rima. 29B was a female individual, 44-49 years old. The height was 158.8 cm; the biomass was 64.8 kg. There was Wells bursitis on the ischial tuberosity on both sides. 29E was a male individual, 45-50 years old. The height was 169.47 cm; the biomass was 70.8 kg. The third and the fourth vertebrae showed Baastrup syndrome (compression of the vertebral spine). There were radiologic signs of deformity on the higher edge of the acetabula and results of frequent sprains of the ankles. 31A was a male individual, 47-54 years old. The height was 178.65 cm; the biomass was 81 kg. The vertebral index showed a heavy overloading in the thoracic lumbar region. There were bony formations under the periosteum on both on the higher and medium facets of the first metatarsus and on the higher and lateral facets of the fifth metatarsus on both sides. As the topography indicates, these small ossifications coincided with the contact points between the back of the foot and parts of the upper shoe. From the osseous remains, in particular from the teeth (central incisors), the DNA was extracted and typed to identify potential family ties among all the subjects. The extraction technique used came from the DNA Promega technique, partially modified by the authors. Stay times of the sample in the extraction buffer were increased and were increased the polymerase chain reaction (PCR) cycles.     

The reliability of forensic osteology--a case in point. Case study

The medico-legal investigation of skeletons is a trans-disciplinary effort by forensic scientists as well as physical anthropologists. The advent of DNA extraction and amplification from bones and teeth has led to the assumption that morphological assessment of skeletal remains might soon become obsolete. But despite the introduction and success of molecular biology, the analysis of skeletal biology will remain an integral part of the identification process. This is due to the fact, that the skeletal record allows relatively fast and accurate inferences about the identity of the victim. Moreover, a standard biological profile may be established to effectively narrow the police investigator's search parameters. The following study demonstrates how skeletal biology may collaborate in the forensic investigation and support DNA fingerprinting evidence.In this case, the information gained from standard morphological methods about the unknown person's sex, age and heritage immediately led the police to suspect, that the remains were that of a young man from Vietnam, who had been missing for 2.5 years. The investigation then quickly shifted to prove the victim's identity via DNA extraction and mtDNA sequence analysis and biostatistical calculations involving questions of kinship [4].     

Forensic aspects of aboriginal skeletal remains in Australia

The assessment of Aboriginal skeletal remains and their distinction from whites is an area of major importance to the Australian forensic pathologist. The Aborigines, the indigenous people of Australia, are a distinct racial group with many characteristic anthropological features. The assessment of race is best made from an examination of cranial traits, 20 which are of value. A characteristic pattern of attrition of the teeth provides a method for establishing a skeleton as pre-European contact Aboriginal or postcontact tribal Aboriginal. The limb proportions of Aborigines differ significantly from other races and provide a useful adjunct to other racial discriminants. Quantifiable sex discriminants in the Aboriginal pelvis and femur differ in their ranges of values from other races. The humerus and shoulder girdle are of no value in making a racial distinction and are of limited value in sexing Aboriginal skeletons. Some pathological changes seen in Aboriginal skeletons are of value in corroborating race. These pathological changes include evulsion of incisor teeth, healed "parry fractures" of the ulnae, tibial squatting facets, treponemal changes, and auditory exostoses. The place and manner of burial together with related artefacts, features, and ecofacts may also provide corroboration.     

Sexual dimorphism in teeth: discriminatory effectiveness of permanent lower canine size observed in a XVIIIth century osteological series

Recent studies have shown that the most dimorphic tooth is the mandibular canine. We have carried out a study on a random sample of 146 skeletons dating from the plague outbreak in Marseilles (1722). We studied 1284 maxillary and 1432 mandibular permanent teeth. Sexual dimorphism was tested on 89 individuals. We selected a set of four dental indices and calculated the dimorphism percentage by ratio expression male/female. Dimorphic ranking was made, by allotting the first rank to the tooth presenting the highest dimorphism and the last rank to the one presenting the lowest ratio. Comparisons of means were made on both sexes (sex determined by post-cranial data) through a Student's test (t-test). We noted that lower canines and lateral incisor are the most interesting teeth in the dimorphic dental determination. The lower index presented the highest relative risk with RR = 1.56 [1.04-2.32]. In 58% of the cases, the lower dental index enabled a correct sex determination (determined on the basis of the post-cranial skeleton). These results showed the existence of a relative dental dimorphism (male > female mesiodistal diameters) with humans. In conclusion, this method, using dental measurements, may be used as an additional technique to determine sex on fragmentary adult skeletons, immature material, missing pieces or ambiguities on post-cranial remains.