关于DNA复制的引物和DNA聚合酶

DNA复制是由DNA聚合酶催化的,反应需要四种脱氧核苷三磷酸和引物-模板;在引物的3′-羟基上,按模板的指令逐个添加脱氧核苷酸,生成碱基序列与模板互补的新DNA。复制时,DNA双链先打开,形成复制叉,随着复制叉的移动完成复制过程。双链DNA的复制是半不连续的,即先导链是连续合成滞后链则为不连续合成;后者先生成若干短片段(冈崎片段),再连在一起。 DNA复制在基因组的加倍、DNA重组以及修复DNA所受损伤等方面都对生命有决定性的作用。     

用于扩增较大片段DNA的PCR方法

用于扩增较大片段ＤＮＡ的ＰＣＲ方法方向东,陈淳（中科院上海生物化学研究所国家分子生物学实验室,上海２０００３１）诸江（上海第二医科大学上海血液学研究所,上海２０００２５）关键词ＤＮＡ扩增,ＰＣＲ自从Ｔａｑ（Ｔｈｅｒｍｕｓａｑｕａｔｉｃｕｓ）ＤＮＡ聚合...     

用于PCR扩增的DNA简易制备法现状

介绍了 6种用于PCR扩增的DNA简易制备法。所得DNA质量符合PCR扩增之用 ,经过试验也可用于系统生物学之研究     

应用引物延预扩增提高微量DNA拷贝数

采用引物延伸预扩增方法,可普遍提高微量模板ＤＮＡ的考贝数,便于进行基因分析时克服标本时少、来源困难的制约,采用常规扩增、检测２４８ｂｐ的ＤＹＺ１片段体系为观察对象,其最小模板量需１．５ｎｇ／２０μｌ体系。以１５个碱基随机寡核苷酸为引物,对最小模板量进行预扩增,再以其产和１／１０为模板,特异扩增ＤＹＺ１片段。进行了相对定量分析,判断源模板ＤＮＡ拷贝数增加的程度。结果１．５ｎｇ男性ＤＮＡ,经随机扩增后     

蛋白质起始的DNA病毒复制机制

DNA聚合酶在DNA合成过程中需要的引物包括RNA引物、DNA自我引物和蛋白质引物3种类型。新DNA链(如冈崎片段)的复制多是在DNA模板上合成一段RNA引物,细小病毒利用其基因组末端的反向末端重复序列(ITRs)自我折叠成DNA引物,而一些DNA、RNA病毒及真菌质粒起始复制反应的引物则是蛋白质。以感染原核生物的噬菌体Phi29和真核DNA病毒腺病毒为例,从复制过程所涉及的蛋白质、对复制原点的识别、复制起始反应、新链的延伸、复制终止过程等方面详细阐述DNA病毒由蛋白质引发的复制机制,并对已商品化的Phi29 DNA聚合酶产品多重置换扩增及单细胞测序等的应用以及基于噬菌体Phi29蛋白质起始的最小复制系统体外扩增异源DNA等最新的应用研究作相关总结介绍。     

一种高效的植物DNA提取和PCR扩增体系建立

报道一种高效简易的植物DNA提取和PCR扩增体系。该体系仅需一步即可提取DNA,扩增时延长PCR预变性时间便能获得较好的目的基因扩增结果。本体系具有较高的实验稳定性和较好的物种适用性,将大大提高植物分子生物学实验和基于分子标记辅助的植物育种实验效率,节省科研成本。     

引物间同源性和裂口对PCR扩增的影响

采用ＰＣ／Ｇｅｎｅ软件对能扩增出特异睡不能扩增的ＰＣＲ引物进行同源性比较,同源间“裂口（ｇａｐｓ）”差数的统计,得出源率小于或等于３５％,“裂口”差数大于或等于３,是获得理想ＰＣＲ扩增效果的关键参数之一的结论,为引物设计提供了一条直观的依据。     

扩增未知序列DNA片段的PCR技术研究进展

1985年,Mullis等人发明了PCR技术,短短十余年间,这一技术得到迅速发展和应用,已由扩增已知基因发展到扩增未知基因,本文旨在介绍利用PCR技术扩增未知序列DNA片段的最新进展及这些技术在基因克隆研究中的应用。     

山蛭基因组DNA的提取及其PCR扩增产物的分析

利用PCR技术从海南山蛭体内分离山蛭素（抗凝血蛋白）基因,首先需获得不带有山蛭体表色素且完整的山蛭基因组DNA,本试验通过结合使用SDS-蛋白酶裂解法和CTAB法,有效的去除了山蛭基因组DNA提取过程中难以去除的色素,得到的基因组DNA保持完整,无降解,以之作为模板,进行PCR扩增,获得一个长度约为237bp的特异性片段,此片段与印度山蛭蛭素的cDNA大小几乎一致。初步表明,这个序列没有内含子,而仅是海南山蛭蛭素的编码序列。     

DNA sequence requirements for replication of polyomavirus DNA in vivo and in vitro.

Cell extracts of FM3A mouse cells replicate polyomavirus (Py) DNA in the presence of immunoaffinity-purified Py large T antigen, deoxynucleoside triphosphates, ATP, and an ATP-generating system. This system was used to examine the effects of mutations within or adjacent to the Py core origin (ori) region in vitro. The analysis of plasmid DNAs containing deletions within the early-gene side of the Py core ori indicated that sequences between nucleotides 41 and 57 define the early boundary of Py DNA replication in vitro. This is consistent with previously published studies on the early-region sequence requirements for Py replication in vivo. Deleting portions of the T-antigen high-affinity binding sites A and B (between nucleotides 57 and 146) on the early-gene side of the core ori led to increased levels of replication in vitro and to normal levels of replication in vivo. Point mutations within the core ori region that abolish Py DNA replication in vivo also reduced replication in vitro. A mutant with a reversed orientation of the Py core ori region replicated in vitro, but to a lesser extent that wild-type Py DNA. Plasmids with deletions on the late-gene side of the core ori, within the enhancer region, that either greatly reduced or virtually abolished Py DNA replication in vivo replicated to levels similar to those of wild-type Py DNA plasmids in vitro. Thus, as has been observed with simian virus 40, DNA sequences needed for Py replication in vivo are different from and more stringent than those required in vitro.     

In vitro HeLa cell DNA synthesis similarity to in vivo replication

An in vitro DNA synthesizing system, consisting of a HeLa cell lysate which incorporated dNTPs into an acid-insoluble, DNase-labile product, was optimized for incorporation per nucleus. Synthesis depended on the presence of all four dNTPs and was linear for about 15 minutes, then slowed and finally stopped after one to two hours at 37 °C. The DNA synthesized in vitro was found to be preferentially attached by covalent linkage to sites which had just been replicated in vivo. DNA fiber autoradiography of DNA labeled in vitro suggests that synthesis occurs by the replicon mechanism proposed for in vivo replication, but at a fork movement rate 50 to 60% of that in vivo.When analyzed on alkaline sucrose gradients, dNTPs appeared to be incorporated by a semidiscontinuous mechanism, with label after brief pulses (10 to 20 s) distributed about equally between a peak of Okazaki fragments and a very heterogeneous distribution of longer DNA strands. Okazaki fragments, which can be initiated in vitro, sedimented in a broad peak averaging 180 nucleotides in length.     

The mechanism of carcinogen-induced DNA amplification: in vivo and in vitro studies.

Exposure to chemical carcinogens provides a means for the enhancement of the frequency of gene amplification and for the facilitation of research into its mechanism(s). Using carcinogen-induced SV40 amplification as a model system it was shown that amplification of the viral sequences occurs via a replication-dependent mode. This process involves overactivation of the origin region and the generation of inverted repeats. Carcinogen-induced enhancement of gene amplification is triggered by cellular factors that could act in trans. An in vitro amplification system, based on extracts from carcinogen-treated cells and SV40 template sequences, was used to further characterize the amplification intermediates. The major products of overreplication in this system consist of sequences derived from the origin region. Our studies suggest that the ability to overreplicate the origin region in vitro derives from the combined action of carcinogen-induced factors that trigger overinitiation, with the inherent inability of Chinese hamster cell extracts to fully replicate large plasmid templates. The newly replicated sequences are not associated with the parental molecule and contain hairpin or stem and loop structures. Based on these findings we propose a novel replicative mechanism for DNA amplification that allows the de novo formation of hairpin structures. According to this model, an obstruction of the replication fork may cause an overturning of the DNA polymerase, followed by a template switch that leads to the use of the newly replicated strand as a template. This mode of replication results in the generation of hairpin structures which can function as precursors for the duplicated inverted repeats which are commonly observed in amplified genomes. This model is supported by our in vitro and in vivo studies. The relevance of this model for the amplification of cellular sequences is discussed.     

Fidelity of replication of bacteriophage phi X174 DNA in vitro and in vivo

Seven different revertants of bacteriophage phi X174am16 (AB5276G leads to T) have been isolated and the nature of the reversions determined by sequencing their DNA. The revertants each differ from am16 by just a single base substitution. These may be distinguished with varying degrees of ease by characteristic temperature sensitivities of growth. This has facilitated the determination of the frequency at which DNA polymerase III catalyses different types of substitution mutations in copying phi X174 DNA in vitro and in vivo. During the replicative form (RF) leads to single-stranded (SS) stage of replication in vitro, four different revertants may be readily produced according to well-defined rate laws on biasing the concentrations of dNTPs. Transversion mutations are found to be formed predominantly by purine x purine mismatching, whilst transitions are formed predominantly by G x T mismatching. The substitutions via G x T and G x A mismatches are estimated to occur at similar frequencies in vivo. The two most common revertants isolated in vivo, however, are not those readily produced during the RF leads to SS stage in vitro but are those produced on purine x purine mismatching in the SS leads to RF stage. The accuracy of the DNA polymerase in vitro appears to be similar to that in this stage in vivo. However, the overall accuracy of the RF leads to SS replication in vivo is more accurate than predicted from the measurements of the accuracy in vitro.     

PCR microfluidic devices for DNA amplification

The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055–1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92–98; Schneegaβ, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101–121; deMello, A.J. DNA amplification: does ‘small’ really mean ‘efficient’? Lab Chip 2001; 1: 24N–29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77–87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28–29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820–825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such as microbial detection and disease diagnosis, based on the DNA/RNA templates used in PCR microfluidics. It is noted, especially, that this review is to help a novice in the field of on-chip PCR amplification to more easily find the original papers, because this review covers almost all of the papers related to on-chip PCR microfluidics.     

Yeast DNA replication in vitro: initiation and elongation events mimic in vivo processes

An enzyme system prepared from Saccharomyces cerevisiae carries out the replication of exogenous yeast plasmid DNA. Replication in vitro mimics that in vivo in that DNA synthesis in extracts of strain cdc8, a temperature-sensitive DNA replication mutant, is thermolabile relative to the wild-type, and in that aphidicolin inhibits replication in vitro. Furthermore, only plasmids containing a functional yeast replicator, ARS, initiate replication at a specific site in vitro. Analysis of replicative intermediates shows that plasmid YRp7, which contains the chromosomal replicator ARS1, initiates bidirectional replication in a 100 bp region within the sequence required for autonomous replication in vivo. Plasmids containing ARS2, another chromosomal replicator, and the ARS region of the endogenous yeast plasmid 2 microns circle give similar results, suggesting that ARS sequences are specific origins of chromosomal replication. Used in conjunction with deletion mapping, the in vitro system allows definition of the minimal sequences required for the initiation of replication.     

DNA amplification using PCR with abutting primers

Molecular Biology - DNA analysis of ñîmplex biological objects (wastewater, soil, archaeological and forensic samples, etc.) is currently of great interest. DNA of these objects is...