Identification of the skeletal remains of a murder victim by DNA analysis

There is considerable anthropological and forensic interest in the possibility of DNA typing skeletal remains. Trace amounts of DNA can be recovered even from 5,500-year-old bones and multicopy human mitochondrial DNA sequences can frequently be amplified from such DNA using the polymerase chain reaction (PCR). But given the sensitivity of PCR, it is very difficult to exclude contaminating material. We now report the successful identification of the 8-year-old skeletal remains of a murder victim, by comparative typing of nuclear microsatellite markers in the remains and in the presumptive parents of the victim. This analysis establishes the authenticity of the bone DNA and the feasibility of bone DNA typing in forensic investigations.     

涉案偶蹄目动物DNA条形码构建

【目的】利用线粒体DNA条形码标记鉴定涉案偶蹄目动物,为解决偶蹄目动物残体、制品的鉴定难题提供技术参考。【方法】对16个偶蹄目动物物种共67份样本,采用试剂盒提取DNA,扩增、测定物种的线粒体DNA的COI基因、Cyt b基因片段;在GenBank上Blast搜索进行同源性分析;利用MEGA 7.0软件分别比较这两个基因片段种内和种间遗传距离,构建系统发育树。【结果】在同源性分析中,西伯利亚狍(Capreolus pygargus)的Cyt b基因片段与GenBank中西伯利亚狍、欧洲狍(Capreolus capreolus)基因序列的同源性均为99.64%~99.82%,该片段不能区分西伯利亚狍与欧洲狍这两个近缘种。其余测序结果与数据库中参考序列一致且相似度较高。对于COI片段,种内遗传距离为0~1.4%,种间遗传距离为1.8%~18.8%。对于Cyt b基因片段种内遗传距离为0~0.5%,种间遗传距离为4.0%~15.8%。两个片段的种内遗传差异均小于种间遗传差异,存在较为明显的条形码间隔。COI和Cyt b基因片段的系统发育树中,同物种的不同个体均能形成具有较高支持度的单分支;不同物种之间界限清晰,每个物种的所有个体均能够形成具有较高支持度的单分支。【结论】基于COI和Cyt b基因片段构建的DNA条形码均可用于对涉案偶蹄目动物的物种鉴定。在物种鉴定中,可采用两个片段联合使用的方式,尤其是对于近缘种的鉴定,以降低错误风险。     

线粒体基因种属鉴定复合扩增体系

针对cyt b基因和ND6基因序列,设计两对引物,以14种常见动物（包括人）生物性检材为对象进行PCR扩增,产物经聚丙烯酰胺凝胶电泳（PAGE）、硝酸银染色进行分析。结果证明,两条电泳条带者为人样本,分别是cyt b基因片段（358 bp）和ND6基因片段（181 bp）;一条电泳条带者为动物来源,是358 bp cyt b基因片段。在37.5μL PCR反应体系中最小检出模板量为0.25 pg基因组DNA。检材在4℃、室温和37℃温度下,经过4个月后均可获得正确的种属区分。高度潮湿环境中放置50 d的检材、形成于水泥地面、墙面、泥土等基质上的血痕,本方法可得到正确区分。由此建立的种属鉴定的线粒体基因复合扩增体系,其检测片段短、灵敏度高、操作简易,适合法医学上微量、降解、陈旧检材的种属判定。     

海南产花鳗鲡细胞色素b基因的克隆及序列分析

线粒体细胞色素b基因是目前研究鱼类分子系统进化的重要分子标记.笔者以日本鳗鲡线粒体基因组序列为模板设计并合成引物进行PCR扩增,并将扩增产物经琼脂糖凝胶电泳和纯化后,克隆到pMD18-T载体、测序,成功得到全长为1 140 bp的海南产花鳗鲡细胞色素b(Cyt b)基因序列.将该基因全长序列递交到Genebank数据库,获得序列登录号为EF690363.用DNA分析软件MEGA2比较了海南产花鳗鲡与递交到GenBank中的8种分布在中国东南沿海、日本海域及南太平洋海域的鳗鲡属(Anguillia)细胞色素b基因的地理差异,并构建了系统进化树.结果显示:日本产和夏威夷产花鳗的地理差异小于海南产花鳗与它们之间的地理差异.     

Minisatellite repeat coding as a digital approach to DNA typing

Most DNA typing systems used in forensic and legal medicine assay allelic length variation at tandem repetitive DNA regions such as minisatellites. A simple alternative approach that displays patterns of variant repeat units along minisatellite alleles is described here. This produces DNA profiles as extraordinarily variable digital sequences appropriate for forensic investigations, including computer databasing, and for analysing allele diversity and the role of recombination in minisatellite instability.     

黄河鮈Cytb基因克隆及其在鮈属中的系统发育

采用聚合酶链式反应克隆黄河鮈线粒体细胞色素b基因 (Cyt b),首次报道了该基因的全长序列(1140bp,FJ904648) ,并与鮈属中其他6个物种的Cyt b 基因进行同源性比较,分析了碱基组成和变异情况,以马口鱼属的马口鱼和鯝属的云南鯝作为外群构建了ML和NJ系统发育树.遗传距离和分子系统学分析表明,黄河鮈与犬首鮈具有较低的遗传距离(6.30%),在系统发育树上两者聚在一起,提示黄河鮈与犬首鮈存在较近的亲缘关系.     

Construction and characterization of a bacterial artificial chromosome library of rice 5460F

Thermo-sensitive genie male sterile (TGMS) rice has a number of desirable characteristics for hybrid rice production. Many studies have demonstrated that the sterility of TGMS rice is controlled by a single recessive gene. It has been mapped for the first time on chromosome 8 and namedtms 1. Several AFLP markers which tightly linked to thetms 1 gene have been identified recently. In order to develop a detailed physical map of thetms1 gene-encompassing region and finally clone thetms1 gene, a bacterial artificial chromosome (BAC) library of rice 5460F (the fertile mutant line of TGMS rice 5460S) using a modified vector pECBAC1 has been constructed. The constructed 5460F BAC library consists of 16 896 clones with an average insert size of 119 kb, which represents about 4.7 times rice haploid genome equivalents. Neither chloroplast nor mitochondrial DNA was detected from the library. The library was screened with three single copy sequence amplified fragment length polymorphism (AFLP) markers which tightly linked totms1 gene as probes and eight positive clones were identified.     

Multiplex amplification of the mammoth mitochondrial genome and the evolution of Elephantidae

In studying the genomes of extinct species, two principal limitations are typically the small quantities of endogenous ancient DNA and its degraded condition, even though products of up to 1,600 base pairs (bp) have been amplified in rare cases. Using small overlapping polymerase chain reaction products, longer stretches of sequences or even whole mitochondrial genomes can be reconstructed, but this approach is limited by the number of amplifications that can be performed from rare samples. Thus, even from well-studied Pleistocene species such as mammoths, ground sloths and cave bears, no DNA sequences of more than about 1,000 bp have been reconstructed. Here we report the complete mitochondrial genome sequence of the Pleistocene woolly mammoth Mammuthus primigenius. We used about 200 mg of bone and a new approach that allows the simultaneous retrieval of multiple sequences from small amounts of degraded DNA. Our phylogenetic analyses show that the mammoth was more closely related to the Asian than to the African elephant. However, the divergence of mammoth, African and Asian elephants occurred over a short time, corresponding to only about 7% of the total length of the phylogenetic tree for the three evolutionary lineages.     

利用12S rRNA基因标记鉴定鱼翅真伪

为检测鱼翅样本真伪及鲨鱼种属,通过提取送检鱼翅、鱼翅羹样本DNA,扩增其线粒体DNA上的用于动物种属鉴定的12S rRNA基因片段,并进行DNA测序和序列分析,测序结果在GenBank上进行BLAST搜索,与数据库中相关物种序列进行同源性分析。结果表明,在送检的23份样本中,18份鱼翅羹样本、2份粉丝状鱼翅、1份翅状鱼翅中均未成功提取到DNA,未扩增出目的基因片段,而从2份鱼翅样本中成功地提取到了基因组总DNA,并成功扩增出了12S rRNA基因片段,这2份样本的12S rRNA基因片段的碱基序列分别与黑边鳍真鲨(Carcharhinus limbatus)、斜锯牙鲨(Rhizoprionodon terraenovae)的同源性达到99%。对鉴定出含有鲨鱼成分的2份样本进行高温泡发处理,高温泡发实验表明,1份样本(22号)有明胶析出。研究证实,12SrRNA基因序列测定技术能准确、快速鉴定待检样本中是否含有鲨鱼成分,结合高温泡发时是否有明胶析出可综合判定鱼翅真伪。     

Analysis of RAPD and mitochondrial 16S rRNA gene sequences from Trichiurus lepturus and Eupleurogrammus muticus in the Yellow Sea

Random amplified polymorphic DNA (RAPD) technique is applied to 12 individuals from each species of the hairtail fishes Trichiurus lepturus and Eupleurogrammus muticus in the Yellow Sea. The percentage of polymorphic sites, degree of genetic polymorphism and genetic distance are compared and the phylogenetic tree is constructed by Neighbor-joining method. The partial mitochondrial 16S rRNA gene is amplified by polymerase chain reaction (PCR) and the PCR products are directly sequenced after being purified. These sequences, together with the homologous sequences of another Trichiuridae species Lepidopus caudatus obtained from GenBank, are used to analyze nucleotide difference and to construct a UPGMA phylogenetic tree by means of biological informatics. Analysis shows: (1) the RAPD technique is a highly sensitive method for investigating genetic diversity in T. lepturus, and E. muticus. T. lepturus exhibits a lower polymorphism and genetic diversity than E. muticus; (2) according to the analysis of the partial mitochondrial 16S rRNA gene sequences, a very low intraspecific variation and considerably high divergence among species were found, which reveals a dual nature of conservatism and variability in mitochondrial 16S rRNA gene; (3) five primers generate the species-specific RAPD sites and these sites can be served as the molecular markers for species identification and (4) it can be proved at DNA variation level that T. lepturus and E. muticus are of two species respectively pertaining to different genera, which supports the Nelson taxonomic conclusion.     

南四湖泥鳅与大鳞副泥鳅CO I基因序列分析

利用PCR-测序技术,对山东省南四湖的泥鳅与大鳞副泥鳅CO I(线粒体氧化酶)基因约900 bp的片段进行测序和分析。这些CO I基因片段的碱基平均含量AT为55.5%,GC为44.5%;序列同源性分析发现,泥鳅和大鳞副泥鳅种内平均遗传距离分别为0.012和0.001,都小于0.02,与其他鱼类的种间遗传距离都大于0.02。利用种间遗传距离构建系统树表明,泥鳅和大鳞副泥鳅样本各自成支,两支的关系比其他鱼种要近,这与形态学分类结果一致,说明该CO I基因序列可以作为DNA条形码用于物种的鉴定。     

随机扩增多态性DNA技术在生命科学炽的应用

DNA分子标记是DNA水平上遗传变异的直接反映,随机扩增多态性DNA（RAPD）技术,是新发展起来的一种DNA分子标记方法。文中详细阐述了RAPD技术的原理,进一步与限制性片段长度多态性（Restriction FragmentLength Polmorphism,RFLP)技术相比,得出它具有快速,简便和对材料要求不高等特点,最后讨论了RAPD技术在生命科学研究中各个方面的广泛应用,包括种基因组的分子谱图建、系统进化发育以及基因定位研究等。     

DNA遗传标记与绵山羊遗传育种

综述了分子遗传标记限制性片段长度多态、随机扩增多态DNA、DNA指纹、微卫星DNA、线粒体DNA限制性片段长度多态、扩增片段长度多态DNA等技术的原理、遗传特点，及其在绵羊和山羊遗传育种中的研究现状，并对其在绵羊和山羊育种中的应用前景作了展望。     

Amplification of specific DNA sequences correlates with multi-drug resistance in Chinese hamster cells

Mammalian cells selected for resistance to certain cytotoxic drugs frequently develop cross-resistance to a broad spectrum of other drugs unrelated in structure to the original selective agent. This phenomenon constitutes a major problem in cancer chemotherapy. Multi-drug resistance arises from decreased intracellular drug accumulation, apparently due to an alteration of the plasma membrane. The observation of double minute chromosomes or homogeneously staining regions in some of the multi-drug-resistant cell lines suggests that gene amplification underlies this phenomenon. We have used the technique of DNA renaturation in agarose gels to detect, compare and clone amplified DNA sequences in Adriamycin- and colchicine-resistant sublines of Chinese hamster cells. We show that both Adriamycin- and colchicine-resistant cells contain amplified DNA fragments, some of which are amplified in both of these independently derived cell lines. Furthermore, loss of the multi-drug resistance phenotype on growth in the absence of drugs correlates with the loss of amplified DNA. These results strongly suggest that the DNA sequences which are amplified in common in multi-drug-resistant cell lines include the gene(s) responsible for a common mechanism of multi-drug resistance in these cells. We have cloned one of the commonly amplified DNA fragments and show that the degree of amplification of this fragment in the cells correlates with the degree of their drug resistance.     

纳豆激酶基因的克隆及其在大肠杆菌中的表达

纳豆激酶是一种良好的天然蛋白酶类溶栓物质。国内外许多学者对该酶进行了基因工程研究,但在克隆表达过程中出现了许多长短不同的基因片段。本研究通过原产日本的优质纳豆中分离鉴定出高产纳豆杆菌N07并提取该菌株的全基因组;通过PCR手段扩增出能编码纳豆激酶信号肽,前导肽和成熟肽的前纳豆激酶酶原基因NK1,以及能编码纳豆激酶成熟肽的纳豆激酶基因NK2,构建了纳豆激酶基因的表达载体pET30a-NK1和pET30a-NK2,转化E.coli BL21后在大肠杆菌中表达,并进行了活性分析。结果发现,纳豆激酶酶原基因片段NK1能成功表达出有活性的分泌型纳豆激酶;而纳豆激酶基因片段NK2的表达产物为无活性的包涵体。在对NK1和NK2的比较研究后可知,纳豆激酶酶原基因片段NK1能在大肠杆菌中很好的分泌表达,这将为纳豆激酶基因工程的深入研究奠定基础。     

葡萄卷叶伴随病毒Ⅲ宁夏分离物外壳蛋白基因的克隆及序列分析

用酶联免疫吸附法对宁夏贺兰山东麓地区不同葡萄品种进行卷叶伴随病毒Ⅲ(GLRaVⅢ)的测定,检测率为37.1%,与田间自然发病率调查结果基本一致.设计GLRaVⅢ外壳蛋白(CP)基因序列引物对,进行RT-PCR扩增,获得1.1 kb的特异片段.其中,CP基因长942 bp,推导的编码蛋白含有313个氨基酸.通过对GLRaVⅢCP基因同源性比较,结果表明,GLRaVⅢ宁夏分离物的CP基因与四川分离物SL-10 CP基因的亲缘关系最近,核苷酸和所编码的氨基酸序列同源性分别为98.8%和98.1%;与巴西分离物PET-4和智利分离物C1-817 CP基因亲缘关系较远,核苷酸序列同源性低于95.0%,所编码的氨基酸序列同源性低于97.0%,认为GLRaVⅢ株系间的CP基因存在差异.     

Conformational mutations in human mitochondrial DNA

Variation in the human mitochondrial DNA (mtDNA) sequence has been extensively analysed using restriction fragment length polymorphisms (RFLPs). MtDNA RFLPs have previously been attributed to nucleotide changes within restriction endonuclease recognition sites or to small insertion-deletion mutations. We now report that RFLPs detected by polyacrylamide gel electrophoresis can also result from single nucleotide substitutions which alter the mobility of small- to medium-sized restriction fragments that incorporate the sequence. We have defined the mutation responsible at two loci and have identified several possible additional loci. When screening human mtDNAs with multiple restriction endonucleases, such mutations can be misidentified as insertion-deletion mutations or counted as multiple polymorphic restriction sites. This can lead to errors in constructing restriction maps and estimating sequence diversity.     

四川黑熊线粒体12S和16S rRNA基因的克隆及序列分析

利用哺乳动物线粒体基因的保守序列设计引物,采用PCR方法,首次从亚洲黑熊四川亚种（Ursus thibetanus mupinensis）的肌肉组织总DNA中扩增出了线粒体12S rRNA和16S rRNA基因并进行了序列测定及分析.结果表明,四川黑熊12S rRNA基因长965 bp;16S rRNA基因长1 580 bp.通过进一步的序列分析表明,四川黑熊的12S rRNA和16S rRNA基因有较高的进化速率,与美洲黑熊、棕熊、北极熊、眼镜熊及大熊猫的相应基因相比较有较大差异,其中与美洲黑熊的同源性相对较高.     

A polymorphic DNA marker linked to cystic fibrosis is located on chromosome 7

Although cystic fibrosis (CF) is among the most common inherited diseases in Caucasian populations, the basic biochemical defect is not yet known. CF is inherited as an autosomal recessive trait apparently due to mutations in a single gene, whence the efforts made to identify the genetic locus responsible by linkage studies. Two markers have recently been identified that are genetically linked to CF: one is a genetic variation in serum level of activity of the enzyme paraoxonase, and the other is a restriction fragment length polymorphism (RFLP) identified with a randomly isolated DNA probe. We report here that the genetic locus DOCRI-917 defined by the cloned DNA probe is located on chromosome 7.