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.     

粪便DNA提取及检验

目的　研究人类粪便DNA的提取和检验方法。方法　 8人份粪便样本 ,磁珠法提取DNA后 ,进行STR复合扩增和mtDNAHVI区测序分析。结果　用 2种方法提取的粪便DNA ,STR复合扩增检验均未获成功 ;方法1提取的粪便DNA有 6个样本、方法 2有 7个样本获得了清晰可读的mtDNAHVI区序列 ,并与唾液对照样本DNA的序列完全一致。结论　用本文建立的方法提取粪便DNA ,不适于STR分析 ,可通过mtDNA测序分析进行检验。     

中国朝鲜族线粒体DNA编码区序列多态性

目的调查中国朝鲜族群体线粒体DNA（mtDNA）编码区内5个部位 3954～4506nt、5218～5974nt、7942～871Int、10296～10653nt 及14496～14867nt的序列多态性。方法采用PCR产物直接测序方法,对212名中国朝鲜族（吉林省延边地区）无关个体进行序列多态性变化和单倍型分布调查。结果在212名无关个体中,共分出148种单倍型。遗传变异度为0．9931,耦合概率为0．0116。测序结果与Anderson标准序列比较,共检测出109个变异位点,其中79个已收录于MITOMAP。结论mtDNA编码区多态性联合应用可以提高mtDNA的个体识别能力。町为相关遗传学研究提供基础数据资料。     

基于Ion Torrent PGM~(TM)测序系统的人mtDNA全测序分析

目的应用Ion Torrent PGM~(TM)测序系统对人线粒体DNA(mitochondria DNA,mtDNA)全序列进行分析检测,研究不同组织间mt DNA序列差异情况。方法通过法医尸体检验采集6名无关个体的组织样本,包括胸腔血液、头发、肋软骨、指甲、骨骼肌和口腔上皮。使用4对引物对线粒体全序列进行扩增,应用Ion Shear~(TM)Plus Reagents试剂盒和Ion Plus Fragment Library试剂盒等构建文库,并在Ion Torrent PGM~(TM)测序系统上进行线粒体基因组全序列测序,并针对异质性位点和在HVⅠ区域突变位点,进行Sanger测序验证。结果所有样本的全基因组mtDNA都扩增成功,6名无关个体分属于6种不同的单倍型,同一个体不同组织之间mtDNA存在异质性差异。异质性位点和HVⅠ区域突变位点采用Sanger测序结果均得到验证。通过Kappa统计方法进行一致性检验后发现,相同个体不同组织的mtDNA序列检验结果仍具有较好的一致性。结论本研究所采用的人线粒体基因组全序列的测序检验方法,可以检测出同一个体不同组织间mtDNA的异质性差异,该差异具有较高的一致性,该结果对mtDNA在法庭科学中的应用具有指导作用。     

人类线粒体DNA的异质型

研究中国人群中线粒体 D 环区不同组织间的差别,用 PCR-测序方法对53个无关个体的毛发与血液样本进行线粒体 DNA 高变区 HVⅠ进行测序分析,通过377测序仪检测,发现1例个体中血液与毛发样本线粒体 HV Ⅰ区15997～16401间存在序列差异。在16235碱基处,毛发样本表现为 T,血液样本为 C/T。结果显示中国人群同一个体不同组织存在线粒体序列差异。     

血细胞线粒体DNA4977缺失与年龄的相关性

目的检测不同年龄组人活体血细胞线粒体DNA4977碱基缺失情况及其与年龄的相关性。方法根据Anderson标准序列设计mtDNA恒定区和mtDNA4977特异缺失区引物,采用实时荧光定量PCR技术,对110份不同年龄组人活体血细胞mtDNA4977缺失水平进行检测。结果在23岁以下个体中未检出mtDNA4977缺失,在大于23岁的被检个体中检测到mtDNA4977缺失,年龄越大,越容易检测到缺失。结论人mtDNA4977缺失与年龄有一定的相关性。     

mtDNA异质性在法医检验中的应用价值

<正> 在一个个体内,各种组织器官及其同一组织的各个线粒体DNA(mitochondrial DNA,mtDNA)序列一致。如果一个个体表现为2种以上的mtDNA序列,则称为该个体mtDNA存在异质性(heteroplas—my)[1,2]。Gill在对疑为俄国沙皇尼古拉二世的遗骸进行mtDNA序列分析时,检测了740个碱基,在16169处发现mtDNA异质性;对其兄弟遗骸测序,     

用PCR和ESI-TOF-MS技术检测线粒体DNA的异质性

目的用PCR和ESI-TOF-MS分型技术检测线粒体DNA（mtDNA）D环高变区,通过碱基组成分析mtDNA的异质性。方法从华东汉族群体选取12名无关个体,用PLEX-ID平台进行mtDNA分型。该平台使用12对引物,对mtDNA高变区1（HVⅠ,引物所跨区域为15893~16451）进行碱基组成分析;使用另外12对引物,对mtDNA高变区2（HVⅡ,引物所跨区域为5~603）进行碱基组成分析,考察mtDNA异质性频率。结果 mtDNA多态性区域的碱基组成信息反映出区段内有无异质性。在高变区Ⅰ的12个区段中,有3个区段表现出多聚C长度异质性：在mtDNA高变区Ⅱ（31~576）的12个区段中,有3个区段检见点异质性,另外5个区域检见Poly C长度异质性。结论群体调查表明,mtDNA的序列异质性多见于高变区Ⅱ的103~267区段,多聚C长度异质性多见于高变区Ⅰ的16124~16201、16157~16201、16182~16250区段和高变区Ⅱ的234~367、431~576区段。将mtDNA标记用于母系关系检验和（或）个体识别时,需要格外留意这些异质性信息,以免结论错误。     

mtDNA 12S rRNA基因序列测定在腐烂动物肌肉样本鉴定中的应用

目的利用mtDNA12SrRNA基因序列测定法对宁波森林公安局查获的一例腐烂动物肌肉样本进行种属鉴定,并探讨该方法在腐烂动物肌肉样本鉴定中的应用价值。方法用酚/氯仿法从腐烂动物肌肉样本中提取出基因组总DNA,再用一通用引物通过PCR技术扩增mtDNA上12SrRNA基因的部分片段并进行序列测定。测序结果在GenBank上进行BLAST搜索,再利用DNAMAN软件进行同源性分析。结果扩增产物序列与东北虎的线粒体DNA的12SrRNA基因序列的部分片段的同源性高达99.9%。结论该动物样本为东北虎,本研究所用方法在野生动物案件的样本鉴定中有极高的应用价值。     

线粒体DNA编码区的多态性

目的研究线粒体DNA(mtDNA)编码区单核苷酸多态性,建立mtDNA编码区多态性在法庭科学中应用的理论基础。方法针对mtDNA编码区nt8162-8483以及nt13070-13299两段序列设计引物,应用直接测序技术研究其多态性。结果两对引物扩增片段长分别为322bp和230bp,共检测到21种变异,24种单倍型,基因多样性为0.7511,两个无关个体的偶合概率为0.2564。结论线粒体DNA编码区多态性位点作为线粒体DNA控制区多态性位点的补充,联合应用可以提高线粒体DNA在法医学应用中的个体识别能力。     

Investigating DNA barcodes of plants growing in some areas of Iran with high crime rate: Quercus brantii,Curpressus arizonica,Crataegus pentagyna,Ziziphus Spina-chtista,and Buxus hyrcana

According to criminal botany, the offender unknowingly carries plant samples from the crime scene. Therefore, studying the genetic data of plants native to the crime scene can solve many ambiguities in the criminal files. In this regard, the aim of this study was to investigate the genome of 5 endemic plants in some areas of Iran with high crime rate. Quercus brantii, Curpressus arizonica, Crataegus pentagyna, Ziziphus Spina-chtista, and Buxus hyrcana were assessed using 1 genetic fragment on plastid regions (trnH-psbA) as well as 1 gene on nuclear chromosome called ITS. The alignment of DNA sequences of trnH-psbA and ITS genes was done using BioEdit, Clustal X, and Muscle v4.0 software programs. The phylogenetic analysis was performed on aligned data using Maximum Parsimony (MP) and the Bayesian methods. The Splits Tree v.4.14.4 software program was used for phylogenetic network analysis. Finally, the data combinability test was conducted using the Incongruence Length Difference (ILD) test by PAUP* software program. All data from nrDNA ITS and trnH-psbA sequences were consistent with Information Compatibility Test (ICT) results. Moreover, the nrDNA ITS indicated more resolved relationship than trnH-psbA. The results from MP and Bayesian analyses did not differ significantly between singular and combined forms, except for a slight variance in confidence interval of branches. As the phylogenetic trees provide more thorough and deeper conception of species relations, it is hoped that they would be useful to illuminate some forensic gaps in regions with high crime rates enriched by these plants, not only in Iran, but also in all areas over the world with this vegetation.     

A polymerase chain reaction (PCR) method for sex and species determination with novel controls for deoxyribonucleic acid (DNA) template length.

Human X and Y chromosome alpha-satellite sequences lying within higher order repeats were amplified by the polymerase chain reaction (PCR) in genomic deoxyribonucleic acid (DNA) isolated from blood, bone, and several other tissues and specimens of potential forensic science interest. X and Y sequences could be coamplified under some of the PCR conditions employed. Monomorphic sequences in the 3'-apolipoprotein B gene (designated "H") and in an alpha-satellite higher order repeat on Chromosome 17 (p17H8, D17Z1) were likewise amplified in the specimens. X and Y sequence amplification can provide information about the sex of origin. Amplification of the X, H, and D17Z1 sequences was found to be primate-specific among the common animals tested and can thus provide species of origin information about a specimen. The authors suggest that amplification of X and D17Z1 or H sequences might provide "relaxed" and "stringent" controls for appropriate PCR amplification tests on forensic science specimens. Testing was carried out using PCR protocols that employed Thermophilus aquaticus (Taq) and Thermus flavis (Replinase) thermostable DNA polymerases.     

Determination of the sequence of intersecting lines using Focused Ion Beam/Scanning Electron Microscope

The aim of this study was to verify that the combination of focused ion beam (FIB) and scanning electron microscope/energy‐dispersive X‐ray (SEM/EDX) could be applied to determine the sequence of line crossings. The samples were transferred into FIB/SEM for FIB milling and an imaging operation. EDX was able to explore the chemical components and the corresponding elemental distribution in the intersection. The technique was successful in determining the sequence of heterogeneous line intersections produced using gel pens and red sealing ink with highest success rate (100% correctness). These observations show that the FIB/SEM was the appropriate instrument for an overall examination of document.     

The search engine's dilemma: implied licence to crawl and cache?

A German court rules that web publishers who choose not to usewidely available technical protection measures, such as therobot exclusion protocol (robot.txt), grant an implied licenceto search engines to crawl and cache for the purpose of indexingcontent on the internet.     

Further evidence of the validity of the Suicide Risk Assessment Scale for prisoners

The Suicide Risk Assessment Scale (SRAS; nine items) is useful in correctional settings but needed further validation. First, 44 inmates originally screened as suicidal with the SRAS were evaluated by institutional psychologists according to five criteria: suicidal urgency, risk, appropriateness of referral, need for short- or long-term watch. On the whole, the SRAS was judged to be as effective as a more elaborate test. Second, 242 suicidal and non-suicidal inmates were tested with the SRAS. Their results correlated better with suicidal risk (.71) than with urgency (.50). Receiver operating characteristic (ROC) analysis even showed that the SRAS performed better than a more elaborate test in predicting risk.     

Comparison of the MMPI-2 and Niederhoffer Cynicism Scales for a Sample of police academy trainees

This study compared the Niederhoffer Cynicism Scale, as a less costly alternative, to the MMPI-2 measure of cynicism. Data were collected on both scales from a sample of male and female police academy trainees to compare reliability and construct validity. Subgroup analyses by race and sex yielded comparable Cronbach alpha estimates for minority (.71) and majority (.83) males to MMPI-2 norms. The Niederhoffer reliability values were marginally smaller (.73 to .77) but within the acceptable range. Construct validity correlations between both scales were significant for minority and majority males. However, sample size was too small for both minority and majority females groups to use appropriate statistical techniques to determine reliability or validity. Continued use of the Niederhoffer is recommended for minority and majority males. Author's Note: This paper was presented at the 44^th Annual Meeting of the Western Social Science Association in Albuquerque, NM, in April of 2002.     

In‐Depth Analysis of Computer Memory Acquisition Software for Forensic Purposes

The comparison studies on random access memory (RAM) acquisition tools are either limited in metrics or the selected tools were designed to be executed in older operating systems. Therefore, this study evaluates widely used seven shareware or freeware/open source RAM acquisition forensic tools that are compatible to work with the latest 64‐bit Windows operating systems. These tools' user interface capabilities, platform limitations, reporting capabilities, total execution time, shared and proprietary DLLs, modified registry keys, and invoked files during processing were compared. We observed that Windows Memory Reader and Belkasoft's Live Ram Capturer leaves the least fingerprints in memory when loaded. On the other hand, ProDiscover and FTK Imager perform poor in memory usage, processing time, DLL usage, and not‐wanted artifacts introduced to the system. While Belkasoft's Live Ram Capturer is the fastest to obtain an image of the memory, Pro Discover takes the longest time to do the same job.     

Handedness identification from intertubercular sulcus of the humerus by discriminant function analysis

The accurate determination of skeletal handedness is a critical requirement in physical anthropology and forensic science. To differentiate handedness, discriminant function analysis is applied using the parameters of right and left intertubercular sulci. A total of 200 matched unpaired adult humeri (100 right and 100 left), collected from the Department of Anatomy, were used to measure four parameters of the intertubercular sulcus namely, width (X1), depth (X2), angle of medial wall (X3) and angle of lateral wall (X4) and to note the presence of supratubercular ridge of Meyer (X5). The discriminant function obtained with the significant variables, angle of medial wall (X3), angle of lateral wall (X4) and presence of the supratubercular Meyer (X5) is: Y = 0.093X3 + 0.056X4 + 0.775X5 - 13.46. The accuracy of correct classification using the discriminant function is 89% which is the best starting point for handedness determination approach. The methodology of using discriminant function can be crucial for medicolegal jurisprudence.     

Forensic Comparison of Automotive Aluminum Wheel Fragments Using Synchrotron Radiation X‐ray Fluorescence with 18‐ and 116‐keV Excitation X‐rays

The performance of synchrotron radiation X‐ray fluorescence (SR‐XRF) spectrometry for nondestructive discrimination of small fragments of automotive aluminum wheels was studied. Fragments (< 500 × 500 μm²) of 45 kinds of wheels were first analyzed by scanning electron microscopy–energy dispersive spectroscopy (SEM‐EDS) and then by SR‐XRF. Despite the Mg/Al intensity ratio being a useful identification index, SEM‐EDS was not efficient enough because of the absence of other meaningful indicators of comparison. Conversely, pairwise comparison was conducted and a 92.9% identification was achieved via SR‐XRF using 18‐keV X‐rays. Trace heavy elements in the high‐energy region were detected by SR‐XRF using 116‐keV X‐rays, and an 82.9% identification was obtained. Combined use of 18‐ and 116‐keV X‐rays allowed 98.2% identification. Hence, SR‐XRF is a powerful tool for nondestructive discrimination of automotive aluminum wheels with high precision using trace elements in a wide energy region.