DNA 改组在病毒新型疫苗研究中的应用

DNA改组是一种全新的体外人工进化模式,它改变了传统的进化途径,大大加速了蛋白质的进化进程.该技术自1994年建立以来,在医药和酶工程等领域得到了广泛的应用.本文综述DNA改组在病毒新型疫苗及其相关领域的研究进展.介绍DNA改组的原理和在体外人工进化上的优势、病毒疫苗研究、病毒载体的改造等方面的研究进展,同时对DNA改组中面临的困难也进行了剖析.     

DNA研究历程及其在法医学中的应用

随着现代生物医学的发展,人类基因组图谱的完成,应用生物化学和分子生物学技术研究DNA及基因的结构与功能、表达和调控,基因组与生命形式和生物形态进化的关系,使人类的研究进入“后基因组学”时代。本文论述了DNA的探索历程及其在法医学中的应用研究,并对DNA的研究技术基因工程、DNA测序、DNA指纹、PCR和DNA芯片等不同检测方法和实际应用效果、前景加以评定。     

重组DNA成功的道路

引言重组DNA(这是科学上的术语、又称“基因工程”或“遗传工程”或“基因捻接”)不是一种普通技术的发展,它与DNA序列分析相结合成为一种非常有力的研究工具。经典遗传学和生物化学技术已逐渐不适用于真核细胞和它们复杂的基因组的研究,没有重组DNA技术,要进一步了解高等生物的分子生物学存在着一定的困难,进展也会比较迟缓。重组DNA拯救了这一领域的“老化”。近年来科学家们利用捻接基因技     

DNA条形码研究进展

DNA条形码（ DNA Barcoding ）技术是一种新的物种识别方法,它是分子生物学和生物信息学相结合的产物。在最近几年里,该技术已成为生物分类学中引人注目的研究热点。这一概念认为,类似于商店里使用扫描仪读取条形码,对地球上每一种生物通过快速分析其DNA中的一段基因（线粒体细胞色素c氧化酶Ⅰ亚基, mt COⅠ）加以识别。理论上, DNA条形码已被证明在生物分类鉴定中具有非常重要的作用,并推动了一系列相关学科的发展,但目前不同分类学家对其持的意见也不尽相同。本文综述了DNA条形码技术的产生、发展概况、原理与操作及其在分类中的应用,并概括了DNA条形码在应用于物种分类中可能存在的问题。     

重组DNA技术及其在产前诊断的应用

近年来,分子生物学和分子遗传学发展迅速,重组DNA技术除了作为研究基因结构、排列和调控最有力的手段外,它已给医学巨大的冲击.国外自七十年代末已将该项技术应用于血液学、肿瘤学、内分泌学、免疫学、遗传病和传染病等方面.本文就重组DNA技术的基本概念及其在产前诊断的应用加以综述.     

DNA芯片技术在药物研究中的应用

DNA芯片技术是21世纪生物技术的重要发展,是生命科学与物理学、化学、微电子学以及计算机学等学科相互交叉的一门高新技术。该技术的突出特点在于其高度的并行性、多样化、微型化、自动化,已在生物医学的各个领域显示出了巨大的发展潜力和应用价值。其中药物研究领域是其应用最广泛的领域之一,通过DNA芯片技术可以将药物的生物效应和基因变化密切相联系,从而为药物的研究、开发注入新的生机和活力。     

DNA芯片技术在药物研究中的应用

DNA芯片技术是21世纪生物技术的重要发展，是生命科学与物理学、化学、微电子学以及计算机学等学科相互交叉的一门高新技术。该技术的突出特点在于其高度的并行性、多样化、微型化、自动化，已在生物医学的各个领域显示出了巨大的发展潜力和应用价值。其中药物研究领域是其应用最广泛的领域之一，通过DNA芯片技术可以将药物的生物效应和基因变化密切相联系，从而为药物的研究、开发注入新的生机和活力。     

杀菌蛋白DNA改组的初步研究

目的：运用DNA家族改组方法提高杀菌蛋白（BPI23）的杀菌活性。方法：首先将人、猴、兔BPI23基因的PCR产物等量混合，用DNAase I消化，回收50bp左右的片段，再经无引物和有引物2步PCR反应获得与原基因大小相同的改组分子，将其与载体连接获得改组文库。应用Flp-In^TM定点整合表达系统，将BPI23改组文库插入CHO细胞基因组中1个单拷贝位点，分别检测各个细胞克隆的杀菌活性，从中筛选高活性的BPI23改组分子。结果：经过2轮改组和筛选，共获得4个活性高于人BPI23约4倍的克隆，它们与人BPI23基因有7－13个氨基酸的变化。结论：探索了CHO系统中进行DNA改组和筛选的方法与技术路线，成功进行了杀菌蛋白的改组，并为其它分子的改组提供了借鉴。     

条件性剔除鼠在神经元发育过程中的应用

条件性基因敲除技术是最近发展起来的新技术，常用的敲除系统有环化重组酶/环化重组酶识别的特异性DNA序列系统、成髓细胞瘤基因转录因子重组酶/铁蛋白位点系统、四环素诱导系统、锌指核酸酶技术、转录激活因子样效应因子核酸酶技术、短回文重复序列/短回文重复序列相关基因系统。该技术能够定向地修饰和改造基因，能够在时间和空间上控制基因敲除，如今已发展成为研究脊椎动物成体中基因功能的重要手段，尤其是该技术在小鼠中的应用更是取得了显著的成果。     

重组DNA及其他先进技术在毒理遗传学中的应用

重组DNA等先进研究方法的建立和发展对毒理遗传学产生了巨大影响。目前已能分离单个突变基因,进行结构与功能的详尽分析;确定基因在正常及异常个体中染色体上的位置,深入了解染色体的结构与功能;利用限制性片段长度多态性(RFLP)及其他实验系统迅速地查出单碱基或微小突变的存在并正确地判断突变的性质及位置;重组DNA技术已开始用于癌形成机理的研究以及环境毒物的快速检测;此外,计算机等先进技术也已进入对诱变、致癌剂分子结构分析及环境物质遗传毒性预言等领域。     

The MASA syndrome: A new heritable mental retardation syndrome

Two Mexican-American children of West Texas are described: a brother and sister with a similar pattern of malformation. They have in common a tetrad of Mental retardation, Aphasia, Shuffling gait, and Adducted thumbs. Besides these two sibs, who were available for study, it is known that a total of seven members of this kindred, six males and one female, spanning three generations, have been similarly affected. Although all members of this family come from the same ethnic background, consanguinity has been denied. The mode of inheritance cannot be firmly established at this time. It is in our opinion most compatible with that of an X-linked recessive gene with phenotypic expression in only one female due to unfavorable Lyonization. Other possibilities exist which cannot be entirely excluded. These include an autosomal dominant mutation with incomplete penetrance in at least three obligatory carrier females. Another possible mechanism is that of an autosomal recessive gene, since all members of this pedigree are of the same Mexican ancestry (Fig. 1. II-1, III-3, III-6). Because all affected members exhibit the four cardinal features of this disorder: Mental retardation, Aphasia, Shuffling gait, and Adducted thumbs, wc wish to refer to this heritable condition as the MASA syndrome until the basic defect is further characterized.     

针对大量噬菌体展示短肽序列的生物信息学分析策略的构建

目的 建立一种生物信息学分析预测方法,针对通过噬菌体展示技术获得的大量短肽信息,从中筛选出所需的目的 功能蛋白. 方法 对应用噬菌体展示技术筛选脂多糖刺激的人外周血单核巨噬细胞株(THP-1)细胞获得的1263个七肽序列进行去冗余分析,结合混洗算法和随机排列检验(RPT),统计非冗余七肽序列中特定三肽的丰度及其显著性.利用Clustal W软件对包含丰度具有显著性的三肽的七肽序列进行多序列比对分析,并根据氨基酸的性质,寻找特征性短肽,通过在线BLAST软件与SWISS-PROT蛋白序列数据库进行同源性分析,获得包含这些特征性短肽的已知蛋白,进一步应用SignalP和TMHMM软件预测其中的分泌蛋白和膜蛋白,并最终获得候选蛋白. 结果 得到TNF-α、toll样受体6(TLR-6)等炎性反应相关的细胞因子和膜蛋白及其表位模拟肽,为脓毒症等炎性反应相关疾病的研究和治疗提供依据. 结论 建立了一种生物信息学分析预测方法,该策略的建立为通过噬菌体展示技术筛选得到的短肽序列的分析提供一个全新的平台.     

Topical application of plasmid DNA to mouse and human skin

Gene expression following direct injection of naked plasmid DNA into the skin has been demonstrated in the past. Topical application of plasmid DNA represents an attractive route of gene delivery. If successful, it would have great prospects in skin gene therapy since it is painless and easy to apply. In this study, we analyzed the expression of plasmid DNA in vivo and in vitro following topical application of plasmid DNA in various liposomal spray formulations. Therefore, different concentrations of plasmid DNA expressing enhanced green fluorescent protein (pEGFP-N1) were sprayed onto mouse or human skin once daily for three consecutive days and compared with direct injection. Gene expression was assessed 24 h after the final topical application of various liposomal DNA formulations. The results showed that EGFP mRNA and protein were detectable by RT-PCR and Western blot, respectively. However, epicutaneously applied EGFP plasmid DNA did not lead to microscopically detectable EGFP protein, when assessed by confocal laser microscopy or fluorescence-activated cell sorting in contrast to about 4% of fluorescent keratinocytes following intradermal injection. In an in vivo mouse model, the application of pEGFP-N1 DNA led to the generation of GFP-specific antibodies. These results indicate that topical spray application of pEGFP-N1 liposomal DNA formulations is a suitable method for plasmid DNA delivery to the skin, yielding limited gene expression. This spray method may thus be useful for DNA vaccination. To increase its attractiveness for skin gene therapy, the improvement of topical formulations with enhanced DNA absorption is desirable.     

History of DNA typing for the human MHC

The last twenty years have seen an exponential growth in the application of DNA technology to the field of Histocompatibility and Immunogenetics (H&I). Initially, this was confined to a few research laboratories. However, the development and application of several different DNA methods by many laboratories has led to the situation whereby nearly every H&I laboratory performs some DNA typing for the detection of HLA alleles. It would not be unfair to say that H&I has shown diagnostic laboratories in other disciplines how useful the DNA techniques can be. This review attempts to summarise the history and application of DNA methods in the field of Histocompatibility (Table 1).     

An improved method for the detection of DNA fragmentation.

An application of the Southern blot technique is described which permits the detection of DNA fragmentation due to cell death by apoptosis. DNA fragments were isolated from cell suspensions and tissues, separated on agarose gel, transferred by Southern blot and hybridized with a radiolabeled total cellular DNA probe. The application of this procedure to thymus cell samples, revealed the distinct ladder pattern of DNA fragments in multiples of about 180-200 base pairs, a characteristic feature of DNA fragmentation. In comparison to conventional DNA visualization with ethidium bromide staining, the radiolabeled probe improved the detection of DNA fragments at least eight-fold. This method detects low levels of DNA fragments, as well as physiological tissue DNA fragmentation, while avoiding cell damage due to DNA radiolabeling.     

全基因组扩增技术最新进展及其法医学应用现状

微量模板DNA的检测，是很多领域迫切需要解决的一个难题。因为其DNA量不足，用现有的技术手段常无法检测成功。全基因组扩增技术可对非常微量的DNA进行均衡的扩增而获得大量的DNA，故被认为是目前解决这一难题的一种基本方法，已被广泛用于法医学、单细胞遗传病的诊断及疾病基因的分析等领域研究，并取得了良好的效果。本文对这一技术的最新研究进展及其在法医学方面的应用现状做一综述。     

Invited Review. Imaging of Single DNA Molecule: Applications to High-Resolution Genomic Studies

Single molecule analysis of DNA has revealed new insights into its structural and physical properties. The application of new methods for manipulating and visualizing DNA has resulted in important advances in high-resolution physical mapping of the genome and quantitative cytogenetic studies of genomic abnormalities (Lichter 1997). Studies of single molecules of DNA have employed a variety of approaches including electron microscopy, atomic force microscopy, scanning-tunneling microscopy and fluorescence microscopy. A number of new technologies have recently been developed to exploit fluorescence microscopy's full potential for genomic analysis and the fine mapping of subtle genetic alterations. In the case of the latter application, particular emphasis has been placed on developing new methods for stretching DNA for high-resolution fluorescence in-situ hybridization studies. We have recently described a process called molecular combing according to which single DNA molecules bound by their extremities to a solid surface are uniformly stretched and aligned by a receding air/water interface (Bensimon et al. 1994). In the following, we will review recent developments concerning molecular combing and discuss its current and potential applications for the high-resolution mapping of the human genome, the detection and quantification of subtle genomic imbalances and the positional cloning of disease-related genes.     

细胞核形态和DNA指数分析在星形细胞瘤分级诊断中的应用

目的 探索自动图像分析系统在胶质瘤分级诊断中的应用。方法 采用自行设计组装的MIPS-I型自动图像分析系统对3个级别星形细胞瘤的细胞核形态和DNA指数(倍体)同时进行原位定量测定。结果 在星形细胞瘤分级诊断中，5项反映核形态的参数诊断正确率为56．3％；代表核DNA改变的7项参数诊断正确率为70．8％；综合应用12项参数诊断正确率为75％。结论 细胞核形态和DNA指数分析可作为星形细胞瘤分级诊断的参考指标之一。     

DNA nanowire translocation phenomena in nanopores

One recent application of nanopores is to use them as detectors and analyzers for fast DNA sequencing. To better understand the DNA electrokinetic transport through a nanopore, a hydrodynamic model is developed to investigate the flow field, the resistive forces acting on the DNA, the DNA velocity and the ionic current through the nanopore. The numerical results reveal the relation between the DNA velocity and various parameters such as nanopore surface charge and solution concentration. The model is validated by comparing the numerical results with the experimental data for both DNA velocity and ionic current through the nanopore.