Taq酶体外合成DNA时模板错位机制的探讨

利用Taq DNA聚合酶体外合成DNA过程中,当反应体系中缺少与模板链互补配对的dNTP底物时,产物合成并不会在底物缺失位点处终止,聚合反应继续进行.为研究此复制缺陷现象,设计一系列模板用于DNA体外酶促合成.除了已知的碱基错配机制,笔者发现存在另一种"模板错位"机制,即模板中与底物非Watson-Crick互补配对的碱基位点首先进行收缩滑动,形成模板bulge结构后再继续进行酶促合成反应.这项研究有助于提高DNA样品合成保真度以及继续深入探索体外DNA合成的详细机制.     

酶法中碳酸甘油单酯的合成

研究了在无溶剂体系中酶促混合中碳酸(辛／癸酸混合物)与甘油合成中碳酸甘油单酯．对8种脂肪酶进行筛选，选定固定化酶LRI为催化剂，以中碳酸转化率为考察指标探讨得到最适酶促反应条件：温度范围为57～60℃，甘油初始含水量12％(质量分数)，n(中碳酸)：n(甘油)=1：1．1、每克酸加酶量为100U，闭口反应2～4h后开口自发脱水。     

四种昆虫病毒基因组DNA限制性内切酶图谱分析

应用限制性内切酶图谱分析,结果ＤＮＡ单分子层和Ｓｏｕｔｈｅｒｎ杂交方法,对黄地老虎颗粒体病毒,甘兰菜粉蝶颗粒体病毒,三叶草夜蛾颗粒体病毒和荨麻蛱蝶核型多角体病毒等四种杆状病毒提取基因组ＤＮＡ用９处限制性内切酶酶切,得到不同的酶切图谱使其在基因型上进行区分,通过ＤＮＡ单分子展层发现其核酸链都呈闭环和超螺旋构型,用＾３２Ｐ标记ＡｕＮＰＶ与ＧＶ杂交,没有发现与ＧＶ的同源性。     

木麻黄小板基因组DNA的快速提取研究

提取介质中加入２．５％β－巯基乙，能有效却除次生物质对林黄基因组ＤＮＡ提取的影响。用新方法所提ＤＮＡ产率比用前人方法提出的高出０．８倍左右，鉴定２结果表明：鲜小枝、干小枝的ＤＮＡ样品皆为４８ｋｂ左右，适于限制性酶切和ＲＡＰＤ反应；同时发现同一株树的鲜小板、干小枝，基因组ＤＮＡ ＲＡＰＤ扩增可得到相同的产物。     

ApiIa抗菌肽基因的化学合成,克隆及其在酵母中的表达

用固相亚磷酰胺法合成了ＡｐｉＩａ抗菌肽基因,全长为８０个核苷酸。它被分成４个寡核苷酸片段,分别在ＤＮＡ合成仪上的合成经分离纯化后的寡核苷酸片段经酶促连接,然后被克隆到分泌型载体质粒ｐＡＦＤ１０１上,经限制酶酶切,ＰＣＲ模板检测以及双链ＤＮＡ序列分析检测,证明合成的ａｐｉＩａ基因和设计的完全一致。     

人ZP3B-细胞表位嵌合肽DNA的设计和合成

目的：设计和合成适合在昆虫细胞中表达的一个人ＺＰ３ Ｂ细胞表位的ＤＮＡ序列。方法：根据国外的研究基础，设计由人ＺＰ３的Ｂ细胞表位肽段和外源Ｔｈ细胞表位肽段串联而成的４５肽嵌合肽序列，并根据昆虫细胞对密码子的偏爱性设计出该嵌合肽的ＤＮＡ序列。然后人工合成该嵌合肽的ＤＮＡ链，并克隆到ＰＵＣ１８载体上。结果：通过酶切分析和测序证明，合成的ＤＮＡ与所设计的嵌合肽ＤＮＡ序列一致。结论：按设计合成了人ＺＰ３的     

维生素C或H2O2系统中HO对DNA的损伤作用

采用紫外分光光度法，ＴＢＡ法，琼脂糖凝胶电泳法检测维生素Ｃ或Ｈ２Ｏ２体系对ＤＮＡ的作用，发现ＤＮＡ在２６０ｎｍ处吸光值下降，检测到ＤＮＡ的脱氧核糖损伤，观察到ＤＮＡ链降解，痕量的过渡金属离子Ｆｅ＾２＋等能加速上述反应的进行，各种ＨＯ清除剂可抑制上述反应。表明在上述两类反应体系中产生了ＨＯ，是ＨＯ破坏ＤＮＡ的碱基和脱氧核糖，降解ＤＮＡ链。     

DNA芯片技术及其农业应用

 ＤＮＡ芯片（ＤＮＡｃｈｉｐ）是生物芯片（ｂｉｏ-ｃｈｉｐ）的一种 ,它有多种同义词 :基因芯片（ｇｅｎｅｃｈｉｐ）、ＤＮＡ显微芯片（ＤＮＡｍｉｃｒｏｃｈｉｐ）、ＤＮＡ阵列（ＤＮＡａｒｒａｙ）、ＤＮＡ显微阵列（ＤＮＡｍｉｃｒｏａｒｒａｙ） ;另外 ,由于ＤＮＡ芯片是一种寡核苷酸 ,所以也称为寡核苷酸阵列（ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅａｒｒａｙ）或寡核苷酸芯片（ｏｌｉｇｏｎｕｃｌｅｏｔｉｄｅｃｈｉｐ）［１］。显微阵列分析源于固体表面的生化试验。芯片技术就是利用高密度的分子阵列分析方法来检测生化样品 ,ＤＮＡ或ＲＮＡ混合物用酶促方法加上核苷酸作为标记形成报告标签 ,再与显微阵列进行杂交。     

人血清中真核复制起始区DNA（Ors12DNA）结合蛋白的鉴定和纯化

利用含有真核细胞复制起始区（ＯｒｉｇｉｎｏｆＤＮＡＲｅｐｌｉｃａｔｉｏｎ）的Ｏｒｓ１２ＤＮＡ作为探针，检测了人血清中与Ｏｒｓ１２ＤＮＡ特异结合的蛋白质。凝胶电泳迁移率改变实验表明人血清中存在特异Ｏｒｓ１２ＤＮＡ结合蛋白。实验还对影响Ｏｒｓ１２ＤＮＡ与蛋白质最适宜结合的各种因素进行了研究。苯酚处理反应体系及限制性内切酶的酶切位点保护实验都证实了Ｏｒｓ１２ＤＮＡ－蛋白质复合物的存在。利用硫酸铵盐析及ＤＮＡ亲和层析，对血清中的Ｏｒｓ１２ＤＮＡ结合蛋白进行了部分纯化，ＳＤＳ－聚丙烯酰胺凝胶电泳结果显示，Ｏｒｓ１２ＤＮＡ结合蛋白为一组非均一的蛋白质，亚基分子量分别为６４ｋＤ、４４ｋＤ和２４ｋＤ。     

栽培高粱叶绿体DNA的酶切片段分析

实验选取了具优良性状的高粱（Ｓｏｒｇｈｕｍｂｉｃｏｌｏｒ）４品种、两细胞雄性不育系,采用改进的高盐低ＰＨ值法对其叶绿体ＤＮＡ进行提取,纯化、限制性内切酶酶切分析。结果表明,改进的高盐低ＰＨ值法无论在ＤＮＡ的得主及质量上都有一定的提高,足以满足ＲＦＬＰ或其他酶切方法的需要;高粱栽培品种间的叶绿体ＤＮＡ变异很少,反映了各族间极其相近的亲缘关系,在可育胞质品种与胞雄性不育系的叶绿体ＤＮＡ之间明显存在两类     

DNA的不同提取方法对转基因产品定值的影响

国内外非常重视转基因产品的检测工作,主要依赖于DNA的PCR方法进行检测,其检测结果可靠、稳定、灵敏度较高。尤其在转基因产品的定值实验中,荧光定量PCR的应用起到重要的作用,但是DNA的质量是保证荧光定量PCR定值准确的关键。实验以100%的克螟稻为材料,用CTAB法、商品化的Promega、Qiagen以及TIANGEN提取试剂盒,分别提取克螟稻基因组DNA,经过核酸蛋白分析仪初步测定浓度、纯度以及离子浓度后,分别以水稻内源基因PLD和克螟稻特异性转化体引物及探针实施荧光定量PCR实验,以100%克螟稻为盲样实施定值,来确定一种更适合荧光定量PCR定值实验的DNA提取方法,最终确定Qiagen和TIANGEN提取试剂盒满足荧光定量PCR对100%的克螟稻标准物质定值实验的要求。     

AP－PCR在肿瘤DNA多态性研究中的应用

首次将ＡＰ－ＰＣＲ应用于ＤＮＡ多态性研究．研究ＡＰ－ＰＣＲ的反应程度，找到实现稳定扩增的适宜条件，并发现模板ＤＮＡ浓度的变化在相当范围内不会改变扩增图谱；对肿瘤、瘤周组织以及正常组织进行分析，在１１个引物中发现２个引物的扩增结果是多态的．初步结论：多态现象与癌变相关．     

Fidelity of DNA replication catalysed in vitro on a natural DNA template by the T4 bacteriophage multi-enzyme complex

More than 50 copies of a phi X174 DNA template can be made in 60 min in an in vitro DNA replication system consisting of seven purfied replication proteins isolated from T4 bacteriophage-infected cells. By transfecting with the DNA products and assaying for the reversion of specific amber mutants, the high degree of base-pairing fidelity in this system is revealed; the in vitro system is also shown to respond to the mutagenic effect of Mn2+ and to display strong base-pair context effects on fidelity, as expected from in vivo studies.     

Mechanism of oxidative damage to DNA by Fe-loaded MCM-41 irradiated with visible light

The mechanism of oxidative damage to deoxyribonucleic acid (DNA) by iron-containing mesoporous molecular sieves (MCM-41) irradiated with visible light was elucidated. Fe-loaded MCM-41 (Fe/MCM-41) was used as a photocatalyst and the damage to calf thymus DNA caused by hydrogen peroxide (H2O2) was studied. The damage and extent of oxidation of DNA were measured by high-performance liquid chromatography (HPLC) and intermediate products were detected by HPLC/electrospray ionization tandem mass spectrometry. Electron spin resonance was used to detect changes in reactive oxygen species and peroxidase catalytic spectrophotometry was used to determine the concentration of H2O2. The results indicated that Fe/MCM-41 efficiently activated H2O2 in solution at pH 4.0-8.0 under irradiation with visible light. The photocatalytic system degraded DNA most effectively at pH 5.0-6.0 but also operated at pH 8.0. At pH 4.2, the degree of DNA damage reached 25.65% after 5 h and the kinetic constant was 5.89×10 2 min 1. Damage to DNA was predominantly caused by hydroxyl radicals generated in the system. The mechanism of DNA damage is of potential concern to human health because it can occur in neutral solutions irradiated by visible light.     

DNA media storage

In 1994, University of Southern California computer scientist Dr. Leonard Adleman solved the Hamiltonian path problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of multiple sequence alignment combined with intelligent heuristics, the most probabilistic file contents can be determined with minimal errors.     

1,3-二甲基金刚烷热解和燃烧的ReaxFF反应动力学模拟

采用基于ReaxFF反应力场的分子动力学方法研究了1,3-二甲基金刚烷(1,3-DMA)在不同温度和质量浓度下的热解和燃烧反应.结果表明,1,3-DMA热解反应开环方式有5种,产物主要为H2,CH4,C2H2,C2H4,C3H4,C3H6,C4H6,观察到H2的生成方式有两种.1,3-DMA燃烧反应主要产物是H2O和CHO类小分子,观察到H2O的生成方式有2种.同时研究了影响热解和燃烧反应速率的因素,温度越高、反应物质量浓度越大热解和燃烧反应速率越大.用ReaxFF动力学方法模拟所得到的结果与实际实验结果一致.     

DNA media storage

In 1994, University of Southern California computer scientist, Dr. Leonard Adleman solved the Hamiltonian path problem using DNA as a computational mechanism. He proved the principle that DNA computing could be used to solve computationally complex problems. Because of the limitations in discovery time, resource requirements, and sequence mismatches, DNA computing has not yet become a commonly accepted practice. However, advancements are continually being discovered that are evolving the field of DNA computing. Practical applications of DNA are not restricted to computation alone. This research presents a novel approach in which DNA could be used as a means of storing files. Through the use of multiple sequence alignment combined with intelligent heu- ristics, the most probabilistic file contents can be determined with minimal errors.     

Frequent chromosomal translocations induced by DNA double-strand breaks

The faithful repair of DNA damage such as chromosomal double-strand breaks (DSBs) is crucial for genomic integrity. Aberrant repair of these lesions can result in chromosomal rearrangements, including translocations, which are associated with numerous tumours. Models predict that some translocations arise from DSB-induced recombination in differentiating lymphoid cell types or from aberrant repair of DNA damage induced by irradiation or other agents; however, a genetic system to study the aetiology of these events has been lacking. Here we use a mouse embryonic stem cell system to examine the role of DNA damage on the formation of translocations. We find that two DSBs, each on different chromosomes, are sufficient to promote frequent reciprocal translocations. The results are in striking contrast with interchromosomal repair of a single DSB in an analogous system in which translocations are not recovered. Thus, while interchromosomal DNA repair does not result in genome instability per se, the presence of two DSBs in a single cell can alter the spectrum of repair products that are recovered.     

Construction of single-chromosome DNA library fromLilium regale Wilson

A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA fromLilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0.5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300–2500 base pairs (bp) with predominant fragments at about 1000 bp. Southern blot analysis confirmed that the PCR products originated from the genome ofLilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1800 bp, with an average of780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).     

芦丁合铁（Ⅲ）的合成、表征及其与DNA相互作用研究

以芦丁和硫酸铁为原料在40℃和pH弱酸性条件下合成芦丁合铁配合物，用等摩尔连续变化法和差热分析法确定配合物的组成为Fe（Rutin）2·5H20；通过UV和IR对配合物的结构进行了表征，该配合物在416nm处有最大吸收，在620．94cm-1处出现Fe-0键的特征吸收峰；运用吸收光谱、荧光发射光谱等方法研究了配合物与DNA的作用机制，并计算出键合常数为67．57mL／mg。实验结果表明，芦丁合铁与DNA以嵌入模式结合。