Multiplex amplification of mitochondrial DNA for human and species identification in forensic evaluation

We describe a method combining in a single-round polymerase chain reaction amplifications of both cytochrome b and hypervariable D-loop mitochondrial DNA allowing species determination and individual human identification. Following the amplification step, amplicons are first screened on an agarose gel. The presence of only one band indicates that the sample is nonhuman, while the presence of two bands indicates a human origin. Subsequent DNA sequencing of the hypervariable D-loop region DNA allows for individual human identification as the presence of cytochrome b fragment does not interfere with the analysis. Similarly, further species determination on the basis of the phylogenetically variable cytochrome b gene is possible by sequencing of the cytochrome b DNA fragment.     

Polymorphism of structural genes of human mitochondrial DNA for forensic medical personal identification

An experimental study is described, which deals with polymorphism of nucleotide sequences of three structural genes of mitochondrial genome, i.e. of the 3d, small 4th, and 6th subunits of the NADN dehydrogenase complex (ND3, ND4L and ND6) sampled from Russian population. The genetic primary structure was analyzed in 63 unrelated individuals. The investigated locuses were shown to possess a pronounced polymorphism. A total of 19 polymorphic positions were detected in the ND3, ND4L and ND6 gene region within the studied sampling. Besides, a possibility is demonstrated in the paper that the mtDNA structural genes can be used as additional identification markers in the forensic experimental typing of the mtDNA control region.     

Application of mitochondrial DNA sequence analysis in the forensic identification of Chinese sika deer subspecies

As a direct and indirect consequence of human activities, only two subspecies, Cervus nippon sinchuanicus and Cervus nippon kopschi, currently subsist in the wild of China. However, a large population of Cervus nippon hortulorum and Cervus nippon nippon is raised in order to gain deer parts for Chinese traditional medicine. According to Chinese Wild Animal Conservation Law, hunting, capturing and trading of the wild sika deer are strictly banned, however, raising and trading of the domestic individual are permitted. Thus, it is very necessary to identify the subspecies of sika deer in China in forensic tests. In our study, we used mitochondrial DNA control region sequence analysis and phylogenetic analysis to identify the subspecies of sika deer. Mitochondrial DNA control region sequences analysis revealed that two haplotypes came from the unknown samples. One is the same as the haplotype that came from the samples of wild population of C. n. kopschi. Phylogenetic analysis indicated that the two haplotypes of unknown samples clustered with the haplotypes of C. n. kopschi, and had significant difference from the haplotypes of the other subspecies. These results together revealed that the unknown samples came from two individuals that belong to the wild population of C. n. kopschi living in the Qinglingfeng State Natural Reserve of Zhejiang province. Therefore, the results provide forensic evidence of illegal wild animal hunting.     

纳米科技与法医DNA检验

综述了纳米科技进步将对法医DNA检验产生的深远影响,对从DNA提取到基因芯片研究等多个未来研究的新方向进行了探讨。     

线粒体16srRNA和ND4基因在种属鉴定中的应用研究

目的构建一种用于种属鉴定的线粒体DNA(m tDNA)16 srRNA和ND4基因荧光标记复合扩增检测体系。方法利用引物设计软件(Prim er 5)对两个m tDNA序列ND4基因和16 srRNA基因设计两对引物,每对引物中的一条在5’端标记荧光素(6-FAM)。按传统复合扩增技术建立复合扩增体系,用AB I PR ISM 310基因分析仪对产物进行分析。结果人类DNA扩增产物出现两个峰,片段大小分别为110bp的人类特异片段和149bp的人与动物共有片段,而动物DNA扩增产物出现一个峰,片段大小为149bp。对30个实验室存放5~15年的陈旧人血痕也能明确判断其种属来源。结论该体系可以明确区分人源性生物检材与其它常见动物样本,对实验室长期存放的陈旧检材也具有较好的检测能力。     

Implications for DNA identification arising from an analysis of Australian forensic databases

Previous analyses of Australian samples have suggested that populations of the same broad racial group (Caucasian, Asian, Aboriginal) tend to be genetically similar across states. This suggests that a single national Australian database for each such group may be feasible, which would greatly facilitate casework. We have investigated samples drawn from each of these groups in different Australian states, and have quantified the genetic homogeneity across states within each racial group in terms of the "coancestry coefficient" F(ST). In accord with earlier results, we find that F(ST) values, as estimated from these data, are very small for Caucasians and Asians, usually <0.5%. We find that "declared" Aborigines (which includes many with partly Aboriginal genetic heritage) are also genetically similar across states, although they display some differentiation from a "pure" Aboriginal population (almost entirely of Aboriginal genetic heritage).     

人线粒体DNA序列分析在法医学中的应用研究及其进展

综述人线粒体DNA(m tDNA)序列分析在法医学种属鉴别、个体识别,以及个体年龄推断中的应用研究及其进展,展望对m tDNA异质性的研究及建立人m tDNA数据库,并具有重要的法医学实践意义。     

Human blood stain identification and sex determination in dried blood stains using recombinant DNA techniques

DNA was extracted from human and non-primate dried blood stains. Human male and female specimens were readily distinguished by analysis with a Y-chromosome specific DNA probe. Human and non-primate blood stains were also readily differentiated using a repeat sequence (Alu) DNA probe. The potential power of recombinant DNA analysis in forensic science is discussed.     

Nuclear and mitochondrial DNA quantification of various forensic materials

Due to the different types and quality of forensic evidence materials, their DNA content can vary substantially, and particularly low quantities can impact the results in an identification analysis. In this study, the quantity of mitochondrial and nuclear DNA was determined in a variety of materials using a previously described real-time PCR method. DNA quantification in the roots and distal sections of plucked and shed head hairs revealed large variations in DNA content particularly between the root and the shaft of plucked hairs. Also large intra- and inter-individual variations were found among hairs. In addition, DNA content was estimated in samples collected from fingerprints and accessories. The quantification of DNA on various items also displayed large variations, with some materials containing large amounts of nuclear DNA while no detectable nuclear DNA and only limited amounts of mitochondrial DNA were seen in others. Using this sensitive real-time PCR quantification assay, a better understanding was obtained regarding DNA content and variation in commonly analysed forensic evidence materials and this may guide the forensic scientist as to the best molecular biology approach for analysing various forensic evidence materials.     

人类线粒体DNA非编码区的序列分析与法医学应用

一、人类线粒体ＤＮＡ(ｍｔＤＮＡ)序列分析和应用的历史沿革1 981年Ａｎｄｅｒｓｏｎ完成了人类线粒体基因组的全部核苷酸序列的测定 ,并提出人类ｍｔＤＮＡ呈闭合环状 ,总长度为 1 6 5 6 9ｂｐ[1 ] 。在此基础上 ,许多学者致力于分析这一环状小分子ＤＮＡ ,以揭示ｍｔＤＮＡ的序列多态性程度。早期主要采用ＲＦＬＰ技术 ,如Ｇｒｅｅｎｂｅｒｇ等[2 ] 、Ｈｏｒａｉ等[3] 用ＲＦＬＰ技术对人类ｍｔＤＮＡ进行了序列分析 ,结果显示 :人类ｍｔＤＮＡ的序列多态性仅局限于长度约为 1 .1ｋｂ的非编码区 ,称之为Ｄ -Ｌｏｏｐ区 ,其中包含两个长度…     

人类线粒体DNA非编码区的序列分析与法医学应用

一、人类线粒体DNA(mtDNA)序列分析和应用的历史沿革 1981年Anderson完成了人类线粒体基因组的全部核苷酸序列的测定,并提出人类mtDNA呈闭合环状,总长度为16 569bp[1].在此基础上,许多学者致力于分析这一环状小分子DNA,以揭示mtDNA的序列多态性程度.早期主要采用RFLP技术,如Greenberg等[2]、Horai等[3]用RFLP技术对人类mtDNA进行了序列分析,结果显示:人类mtDNA的序列多态性仅局限于长度约为1.1kb的非编码区,称之为D-Loop区,其中包含两个长度各为400bp的高度可变区-HV1和HV2;不同个体的mtDNA存在长度变异和序列变异,结果也提示人类线粒体DNA比核DNA有更高的突变率,为核DNA的5～10倍.甚至某些区域是核DNA的6～17倍.到了90年代,DNA自动测序技术在mtDNA研究上的普及应用,大大促进了研究的发展,不少学者提出人类mtDNA的序列分析可用于法医学个人识别.如Stonking等[4]用SSO杂交技术,Sulivan等[5]和Holland等[6]用直接测序法分别对时间久远(最长达24a)的尸体残骸的mtDNA进行序列分析,并与其可疑母系亲属进行比对,为尸源追踪提供了证据. 国内法医学者也于90年代中期开始了对我国汉族人群的mtDNA D-Loop区的序列进行分析[7,8],并陆续有将mtDNA的序列分析用于法医个人识别的报道,如公安部二所的刘冰等[9]将对脱落毛发的mtDNA嵌套式扩增的方法用于模板量很少的案例的个人识别,获得成功. 二、人类mtDNA序列分析的现状目前对mtDNA序列的分析方法多采用对其PCR产物的自动测序,所用检材包括血液、毛发、皮肤、指甲、骨骼、胎盘等多种组织,仍以Aderson所报道的序列为参考序列.     

Human hair histogenesis for the mitochondrial DNA forensic scientist.

Analysis of mitochondrial DNA (mtDNA) sequence from human hairs has proven to be a valuable complement to traditional hair comparison microscopy in forensic cases when nuclear DNA typing is not possible. However, while much is known about the specialties of hair biology and mtDNA sequence analysis, there has been little correlation of individual information. Hair microscopy and hair embryogenesis are subjects that are sometimes unfamiliar to the forensic DNA scientist. The continual growth and replacement of human hairs involves complex cellular transformation and regeneration events. In turn, the analysis of mtDNA sequence data can involve complex questions of interpretation (e.g., heteroplasmy and the sequence variation it may cause within an individual, or between related individuals. In this paper we review the details of hair developmental histology, including the migration of mitochondria in the growing hair, and the related interpretation issues regarding the analysis of mtDNA data in hair. Macroscopic and microscopic hair specimen classifications are provided as a possible guide to help forensic scientists better associate mtDNA sequence heteroplasmy data with the physical characteristics of a hair. These same hair specimen classifications may also be useful when evaluating the relative success in sequencing different types and/or forms of human hairs. The ultimate goal of this review is to bring the hair microscopist and forensic DNA scientist closer together, as the use of mtDNA sequence analysis continues to expand.     

Polymorphism of the central region of D-loop of mitochondrial DNA and personality identification by forensic medicine methods

Polymorphism of mDNA D-loop central region (CR), positions 16366-0072) was studied versus hypervariable sections (HVS1), positions 16024-16365, and HVS2, positions 00073-00340, for a sample of 71 residents of the Russian Federation. Ten polymorphic positions with 56 nucleotide substitutions, 55 of which are transitions, were detect in the CR section; no insertions or deletions were found there. It was proven as possible to use the mDNA CR locus as an additional identification marker in the forensic-expert of the mDNA control region. The probability of random coincidence (RC) of haplotypes in joint typing of HVS1, HVS2 and CR made 0.0208, which is 1.4-fold less versus the same parameter for haplotypes HVS1/HVS2 (RC = 0.0284).     

A PCR multiplex and database for forensic DNA identification of dogs

Animal-derived trace evidence is a common finding at crime scenes and may provide an important link between victim(s) and suspect(s). A database of 558 dogs of pure and mixed breeds is described and analyzed with two PCR multiplexes of 17 microsatellites. Summary statistics (number of alleles, expected and observed heterozygosity and power of exclusion) are compared between breeds. Marked population substructure in dog breeds indicates significant inbreeding, and the use of a conservative theta value is recommended in likelihood calculations for determining the significance of a DNA match. Evidence is presented that the informativeness of the canine microsatellites, despite inbreeding, is comparable to the human CODIS loci. Two cases utilizing canine DNA typing, State of Washington v. Kenneth Leuluaialii and George Tuilefano and Crown v. Daniel McGowan, illustrate the potential of canine microsatellite markers for forensic investigations.     

Analysis of genetic diversity of the mitochondrial DNA in the forensic medical expert identification of individuals

Spectra of haplotype frequencies were studied for locuses of hypervariable segments 1 and 2 (HVS1 and HVS2), separately for each, and for linked segment HVS1-HVS2. The obtained data were used to determine the values and to evaluate comparatively the discriminating characteristics of the corresponding individualizing systems based on the typing of mtDNA. The system of typing, based on HVS2 (mv = 0.098), was found to possess the least discriminating potential; while the highest information rate is ensured in the analysis of HVS1 (m omega = 0.02) and in the joint analysis of HVS1 and HVS2 (mw = 0.007). The frequency rates of the key haplogroups were estimated within a random sampling of Russian citizens. A random population sampling of Russian citizens was shown not to differ essentially from an ethnically homogeneous population sampling of Russians selected with regard for a genetic diversity and for a spectrum of mitochondrial lines. The results point at the most rational algorithm of examinations in a forensic expert's analysis of mtDNA. The studied sampling can trigger the development of a referential data base designed for conducting, in the Russian Federation, the forensic-medical expert's examinations based on the mtDNA typing.     

Sample collection strategies when building mitochondrial DNA forensic databases

For establishing databases that capture the existing diversity in populations, the sample collection strategy is a determining factor and caution must be taken when choosing the suitable approach. Many researchers choose to restrict the sampling to individuals with inheritance for three generations in a specific geographic location. However, the appropriate database in a forensic context is the one representing the current population. We analyzed mtDNA composition across generations in populations from Colombia, Ecuador, and Paraguay. An overall genetic homogeneity was detected, with statistically significant differences on macrohaplogroup frequencies for few department/regions.