DNA测序技术方法研究及其进展

DNA测序技术发明于20世纪70年代,经过40多年的发展,现已成为分子生物学领域最常用的DNA研究方法之一。本文对第一代和第二代的DNA测序技术进行介绍,着重阐述其原理和发展历程,以及应用的主要领域。在此基础上对第三代DNA测序技术进行比较,为研究人员今后进行相关研究提供参考。     

《DNA指纹法》

《DNA指纹法》(DNA fingerprinting)由Lorne T.Kirby著,1990年Stockton出版社出版,365页。该书是《分子生物学上突破》(Breakthroughs in molecular biology)系列丛书中的一种。这套丛书以其高质量在分子生物学和免疫学上占有一席之地,它以快速出版,向读者提供最新突破,边缘技术和合成,以及分子生物学的主要进展。《DNA指纹法》是种创新技术,它能使科学家去鉴别极微小组织样品,能对动物及植物种群的组成、生殖、进化加以科学研究。在法医学中可用做工具,对犯罪施行精确鉴定,发挥着重要作用。该书出版可     

DNA多态性分析技术研究进展

随着现代分子生物学的迅速发展,DNA多态性技术在很多领域得到了应用,本文简单总结了DNA多态性分析技术的发展,并展望了DNA多态性技术的应用前景。     

直接从土壤中提取DNA的方法

研究微生物的多样性 ,即微生物的种类和数量的多少是评价土壤质量的重要指标。由于土壤微生物种类繁多 ,数量巨大 ,加上土壤中 99%的种类难以通过传统的平板分离技术来进行培养[1],人们必须借助其他技术来解决。近年发展的非培养技术 ,如ＢＩＯＬＯＧ微量板分析技术[2 ]、细胞壁磷脂酸分析技术[3]和分子生物学方法[4 - 6 ],克服了培养的环节 ,对微生物生态学研究产生极大的推动作用。其中分子生物学是应用最广、最有发展潜力的技术。它的主要步骤是通过直接提取土壤中的ＤＮＡ ,经纯化处理后 ,利用合适的引物扩增 1 6ＳｒＲＮＡ基因 ,通过分…     

DNA克隆和组装技术研究进展

DNA克隆和组装技术是重要的分子生物学工具。近年来,随着合成生物学的飞速发展,对大片段DNA元件的快速有效组装就显得尤为关键。同时,各种DNA克隆和组装技术也竞相发展起来。通过对基于非典型酶切连接、PCR、同源重组、单链退火拼接等原理发展起来的各种DNA克隆和组装技术进行综述,为合成生物学的进一步发展提供有效的操作工具。     

DNA序列分析技术及其现状

ＤＮＡ序列分析技术一直是分子生物学领域的一个重要研究课题。“人类基因组计划”的实施有力地推动了高速ＤＮＡ测序技术的发展。本文主要介绍国内外目前流行的经典的测序方法———各种改良的双脱氧链末端终止法,及近年来发展起来的一些全新的ＤＮＡ测序方法     

真菌多样性研究方法进展

真菌广泛存在于自然界,在生态系统中发挥着重要的作用.随着分子生物学技术在微生物多样性研究中的广泛应用以及宏基因组学技术的出现,打破了传统微生物培养法的局限性,人们对于真菌多样性的认识日渐提高.该文主要综述了研究真菌多样性方法的主要发展历程,介绍几种有关真菌多样性研究常用的分子生物学方法,包括基于PCR的克隆文库方法、变性梯度凝胶电泳、末端限制性长度多态性、实时荧光定量PCR、荧光原位杂交、序列标签标记的高通量测序以及宏基因组技术,并且阐述宏基因组学技术在此领域蕴含的巨大发展潜力.     

DNA微阵列在药物研究中的应用

DNA微阵列是传统分子生物学向后基因组学过渡中发展起来的新技术,具有高通量,速度快的优点,并能够在药物和基因之间架起一座桥梁.从基因水平上来解释药物的作用机理,因此在新型药物开发和分析中具有广泛的应用前景.本文着重介绍了该技术的概况和在药物开发和分析中的应用,并提出了该技术在药物开发和分析的前景和展望.     

重组DNA技术已被列入美国大学有关系科的实验课程

重组DNA技术是从分子生物学发展起来的一门崭新的技术,与工农业生产和医学事业关系十分密切。最近美国加利福尼亚大学首次为有关系科的大学生和研究生开设了以重组DNA技术为重要     

流感病毒DNA疫苗的研究进展

流感病毒是一种危害极大的病原体。常见的流感疫苗有灭活疫苗、减毒活疫苗。随着分子生物学技术的发展,流感DNA疫苗成为流感疫苗的一个重要发展方向。常见的流感DNA疫苗有血凝素(HA)、神经氨酸酶(NA)DNA疫苗、核壳蛋白(NP)、膜蛋白(M)DNA疫苗。联合使用细胞因子DNA对流感DNA疫苗的免疫效果有明显加强作用。众多试验表明,流感病毒DNA疫苗效果良好,其保护效果不逊于传统灭活疫苗,具有良好的发展前景。     

Capping DNA with DNA

Twelve classes of deoxyribozymes that promote an ATP-dependent "self-capping" reaction were isolated by in vitro selection from a random-sequence pool of DNA. Each deoxyribozyme catalyzes the transfer of the AMP moiety of ATP to its 5'-terminal phosphate group, thereby forming a 5',5'-pyrophosphate linkage. An identical DNA adenylate structure is generated by the T4 DNA ligase during enzymatic DNA ligation. A 41-nucleotide class 1 deoxyribozyme requires Cu(2+) as a cofactor and adopts a structure that recognizes both the adenine and triphosphate moieties of ATP or dATP. The catalytic efficiency for this DNA, measured at 10(4) M(-1) x min(-1) using either ATP or dATP as substrate, is similar to other catalytic nucleic acids that use small substrates. Chemical probing and site-directed mutagenesis implicate the formation of guanine quartets as critical components of the active structure. The observation of ATP-dependent "self-charging" by DNA suggests that DNA could be made to perform the reactions typically associated with DNA cloning, but without the assistance of protein enzymes.     

Eukaryotic DNA polymerases in DNA replication and DNA repair

DNA polymerases carry out a large variety of synthetic transactions during DNA replication, DNA recombination and DNA repair. Substrates for DNA polymerases vary from single nucleotide gaps to kilobase size gaps and from relatively simple gapped structures to complex replication forks in which two strands need to be replicated simultaneously. Consequently, one would expect the cell to have developed a well-defined set of DNA polymerases with each one uniquely adapted for a specific pathway. And to some degree this turns out to be the case. However, in addition we seem to find a large degree of cross-functionality of DNA polymerases in these different pathways. DNA polymerase α is almost exclusively required for the initiation of DNA replication and the priming of Okazaki fragments during elongation. In most organisms no specific repair role beyond that of checkpoint control has been assigned to this enzyme. DNA polymerase δ functions as a dimer and, therefore, may be responsible for both leading and lagging strand DNA replication. In addition, this enzyme is required for mismatch repair and, together with DNA polymerase ζ, for mutagenesis. The function of DNA polymerase ɛ in DNA replication may be restricted to that of Okazaki fragment maturation. In contrast, either polymerase δ or ɛ suffices for the repair of UV-induced damage. The role of DNA polymerase β in base-excision repair is well established for mammalian systems, but in yeast, DNA polymerase δ appears to fullfill that function. Received: 20 April 1998 / Accepted: 8 May 1998     

DNA topoisomerases and DNA repair

DNA topoisomerases are enzymes that can modify, and may regulate, the topological state of DNA through concerted breaking and rejoining of the DNA strands. They have been believed to be directly involved in DNA excision repair, and perhaps to be required for the control of repair as well. The vicissitudes of this hypothesis provide a noteworthy example of the dangers of interpreting cellular phenomena without genetic information and vice versa.     

DNA knots and DNA supercoiling

Comment on: Witz G, et al. Proc Natl Acad Sci USA 2011; 108:3608-11.     

DNA supercoiling by DNA gyrase

Using purified DNA gyrase to supercoil circular plasmid pBR322 DNA, we examined how the linking number attained at the steady state (‘static head’) varies with the concentrations of ATP and ADP, both in the absence and presence of spermidine. In the absence of spermidine at total adenine nucleotide concentrations between 0.35 and 1.4 mM, the static-head linking number was independent of the sum concentration of ATP and ADP, but depended strongly on the ratio of their concentrations. We established that the same linking number was attained independent of the direction from which the steady state was approached. The decrease in linking number at static head is more extensive when spermidine is present in the incubation, but remains a function of the [ATP]-to-[ADP] ratio. These results are discussed in terms of various kinetic schemes for DNA gyrase. We present one kinetic scheme that accounts for the experimental observations. According to this scheme our experimental results imply that there is significant slip in DNA gyrase when spermidine is absent. It is possible that spermidine acts through adjustment of the degree of coupling of DNA gyrase.     

DNA supercoiling inhibits DNA knotting

Despite the fact that in living cells DNA molecules are long and highly crowded, they are rarely knotted. DNA knotting interferes with the normal functioning of the DNA and, therefore, molecular mechanisms evolved that maintain the knotting and catenation level below that which would be achieved if the DNA segments could pass randomly through each other. Biochemical experiments with torsionally relaxed DNA demonstrated earlier that type II DNA topoisomerases that permit inter- and intramolecular passages between segments of DNA molecules use the energy of ATP hydrolysis to select passages that lead to unknotting rather than to the formation of knots. Using numerical simulations, we identify here another mechanism by which topoisomerases can keep the knotting level low. We observe that DNA supercoiling, such as found in bacterial cells, creates a situation where intramolecular passages leading to knotting are opposed by the free-energy change connected to transitions from unknotted to knotted circular DNA molecules.     

Characterisation of rye B chromosome DNA by DNA/DNA hybridisation

The DNAs of wheat and rye plants with rye B chromosomes have been compared with wheat, rye and oats DNAs by DNA/DNA hybridisation. The presence of DNA from B chromosomes made no significant difference to the proportion of repeated sequence DNA. The repeated sequence fractions of these cereal DNAs were quantitatively divided into eight different groups on the basis of the amount of DNA/DNA hybridisation occurring between the different DNAs. Rye A and B chromosomes contained similar proportions of three of the groups. These results, together with estimates of the thermal stabilities of all the renatured DNA duplexes suggest that rye B chromosome DNA is very similar to rye A chromosome DNA in the proportion and heterogeneity of its repeated sequences.     

DNA topology: dynamic DNA looping

The DNA in repressive loops is often tightly bent. DNA flexibility imposes significant constraints on their topology suggesting that they may exist as perturbations in plectonemic DNA.