基于AFLP分析用吴茱萸叶高质量DNA的提取

目的：研究吴茱萸叶基因组DNA的提取方法,以用于扩增片段长度多态性（AFLP）分析。方法：设计了一种改良CTAB法：以石英砂代替液氮研磨;抽提前用不溶性PVP结合酚形成络合物,然后用缓冲液除去;抽提中加入Vc。将改良CTAB法所提吴茱萸叶DNA与传统的SDS法、CTAB法所提DNA进行比较。利用植物的核糖体DNA（rDNA）保守序列设计引物行PCR扩增鉴定吴茱萸DNA及其质量。并确定提取方法中最佳样本含量和β-巯基乙醇浓度。结果：改良CTAB法提取石虎、疏毛吴茱萸总DNA呈白色,A260/A280为1.721~1.886,DNA分子完整,约20kb左右,PCR扩增条带清晰、明亮,无杂带和脱尾。并确定0.10g为最佳样本量,2.0%为最佳β-ME浓度。结论：石英砂研磨简便、迅速、均匀,该实验所建立的改良CTAB法可有效避免次生代谢物的氧化褐变,是一种小量、快速提取吴茱萸叶DNA优化方法。     

檀香心材总RNA提取方法的比较研究

为筛选檀香心材总RNA提取方法,对5种提取方法进行比较研究,包括Trizol法、改良CTAB法、SDS酸酚法、异硫氰酸胍-CTAB法、异硫氰酸胍-SDS法。结果表明,Trizol法和异硫氰酸胍-CTAB法不能提取出檀香心材总RNA,而SDS酸酚法、改良CTAB法和异硫氰酸胍-SDS法均能提取檀香心材总RNA。SDS酸酚法的A260 nm/A230 nm小于2.0,且RNA产率低,仅为(27.94±1.06)μg g–1,不能满足后续实验要求。而改良CTAB法和异硫氰酸胍-SDS法提取的总RNA带型清晰,完整性好,A260 nm/A280 nm为1.8~2.0,A260 nm/A230 nm大于2.0,RNA产率分别为(79.06±4.22)和(107.00±1.36)μg g–1。分别以改良CTAB法和异硫氰酸胍-SDS法提取的总RNA为模板,通过RT-PCR反应,扩增檀香Actin基因片段,结果二者扩增产物大小相同且条带单一,说明改良CTAB法与异硫氰酸胍-SDS法为檀香心材总RNA提取的较好方法。     

顽拗植物澳洲坚果成熟叶片DNA提取方法比较

目的:针对澳洲坚果成熟叶片中富含多糖、多酚等杂质的特点,建立澳洲坚果成熟叶片中提取高质量 DNA 的方法.方法:采用改良CTAB法和改良SDS法提取澳洲坚果样品的总DNA,并对产物进行紫外、电泳及PCR扩增检测.结果:改良CrAB法的平均产率为13.6μg/g,略低于改良SDS法18.5μg/g,但改良CTAB法可有效去除多糖等杂质,获得的基因组DNA质量高.OD260/OD280均在1.7～1.9之间.进行ISSR扩增可获得清晰、多态性好的条带.结论:改良CrAB法较之改良SDS法更适合于从澳洲坚果成熟叶片中提取高质量DNA.     

应用改良CTAB法提取树莓DNA最佳条件的探索

目的:改良CTAB法提取三种树莓叶DNA最佳条件的探索及检测DNA的完整度。方法:通过改良CTAB法提取树莓叶三个品种的基因组DNA。结果:经检测,所提样品OD260/OD280值在1.6~1.9之间,得率为198~396ng/μl,电泳条带较清晰,无明显拖尾现象;提取最佳条件为水浴时间90min、CTAB抽提液浓度3%时,发现DNA完整性较好、纯度较高,可以满足相关的分子生物学研究需要。结论:该研究可以为树莓叶分子生物学的后续研究的开展奠定实验基础。     

观赏桃ISSR-PCR反应体系的优化

采用改良CTAB法从观赏桃满天红叶片中提取基因组DNA,通过单因素实验探讨了模板DNA、Mg~(2+)、dNTPs和Taq DNA酶等条件对观赏桃ISSR-PCR扩增结果的影响,建立了ISSR-PCR扩增的最佳体系:2.5μl反应体系中包含10×Buffer 2.5μl,模板DNA 40ng,Mg~(2+)浓度2.5mmol/L,引物浓度04 μmol/L,dNTPs浓度0.4mmol/L,Taq DNA酶0.5U.利用所建立的体系对红叶桃、菊花桃和春艳等13份材料进行检验,其结果表明优化后的体系适合观赏桃的ISSR-PCR反应.     

一种高效可直接用于PCR分析的土壤总微生物DNA抽提方法

以CTAB-溶菌酶-蛋白酶K-冻融裂解法直接抽提土壤总微生物的基因组DNA,利用G8000沉淀和纯化DNA.结果表明,该方法是一种简便、有效可直接应用于PCR分析的土壤总微生物基因组DNA的抽提方法.采用含聚乙烯吡咯烷酮(PVP)的缓冲液预洗,添加CaCl₂和BSA,可以去除腐殖酸;用PEG8000沉淀DNA,可以提高DNA质量;采用冻融法破碎细胞,CTAB、溶菌酶和蛋白质酶K共同作用以裂解细胞,可以保证获得大片段的DNA,提高DNA产率.用该方法抽提的七子花林下土壤总微生物DNA产率为9.22 μg·g^-1,A260/A280为1.65,可适用于 PCR扩增及扩增rDNA限制酶切分析(ARDRA)技术,适宜的模板DNA浓度为0.67 ng·μl^-1.快速、有效、可直接用于PCR分析的土壤总微生物DNA提取方法的建立,为大规模的土壤微生物分子生态学研究提供了可能.     

水霉菌总DNA提取方法研究

本实验采用EP管反复冻融和研磨的破壁方式,利用溶菌酶法、CTAB法、改良CTAB法、尿素法、十二烷基硫酸钠(SDS)法等5种方法,分别对5种鱼类致病性水霉菌(寄生水霉、多子水霉、异株水霉及两未定种)的基因组DNA进行提取,并采用紫外分光光度计和ITS区基因(包括5.8S rDNA)PCR扩增对DNA进行了评价.紫外分光光度计检测结果表明,5种方法均可提取到水霉菌DNA,其中改良CTAB法提取的5种水霉菌的DNA产量和质量最高,A260/A280在1.79-1.82之间,浓度为45μg/mL;PCR检测结果表明,只有改良CTAB法提取的DNA全部扩增到明亮、整齐、无拖尾的特异性条带,其他几种方法均存在暗带或无带现象.因此,改良CTAB法可以作为水霉菌DNA提取以开展分子生物学研究的首选方法.     

红曲霉DNA提取及其RAPD-PCR反应体系的建立

采用改进的氯化苄法对红曲霉DNA进行提取纯化,探讨了提取液中EDTA的浓度以及其他因素对提取结果的影响。同时,以该法提取的DNA为模板,采用正交实验优化RAPD分析最佳反应条件。结果表明,当提取液中EDTA浓度为125mmol/l时,所得的红曲霉DNA的质量和数量均较理想,每克红曲霉菌丝体（湿重）能提取到50μg的DNA,分子量约为25kb,以此DNA为模板进行PCR扩增,其最佳反应体系为：Mg^2 2.0mmol/l,dNTPs 0.15mmol/l,Taq 0.05U/μl,模板DNA 1.2ng/μl,随机引物0.36μmol/l,Tris-HCl 10mmol/l pH9.0,KCl 50mmol/l,Nonidet P40 0.1%。另外,通过对比RAN酶消化前后的模板扩增结果证明了本实验酶的消化的必要性,对比了不同预变性时间处理模板对RAPD－PCR扩增的影响,发现不经过预变性的模板扩增结果最理想。     

玉米花粉粒直接PCR技术研究

利用成熟花粉粒制成悬液作为玉米基因组DNA模板直接进行PCR扩散,研究了不同花粉悬液浓度、花粉上清液对RAPD-PCR的影响,结果表明：花粉悬液浓度在4μg/50μl以上,用10碱基随机引物均能扩增出较清晰的PCR条带,且与叶片按改良Guidet法提取的DNA模板扩增的RAPD带型无明显差异,利用花粉悬液能有效地进行基因组DNA变异的RAPD分析和基因SCAR标记的检测。玉米单株花粉量可用于数百次以上的PCR反应,较叶片等植物组织用常规法提取DNA快速、简便、廉价,可有效地应用于以PCR为基础的植物基因组DNA变异,农艺性状的RAPD标记和分子标记辅助育种的研究。     

宽叶缬草DNA的提取及正交优化RAPD反应体系

[目的]确定适合宽叶缬草RAPD分析的DNA提取方法以及建立最佳RAPD反应体系。[方法]比较宽叶缬草基因组DNA的两种提取方法(经典CTAB法、试剂盒法);采用正交设计L16(45),针对Taq DNA聚合酶浓度,d NTP浓度,Mg2+浓度,引物浓度,DNA模板浓度进行RAPD扩增,确立最佳RAPD反应体系。[结果]综合比较,试剂盒法较适合宽叶缬草基因组DNA提取;25μl最适宽叶缬草RAPD反应体系为:2.0 U Taq DNA聚合酶、0.4 mmol/L d NTP、4.0 mmol/L Mg2+、4.0μmol/L随机引物、60 ng模板DNA、2.5μl 10×buffer。[结论]试剂盒DNA提取法和正交优化的反应体系适用于宽叶缬草的RAPD分析,为进一步研究黔产宽叶缬草药材遗传多样性奠定了基础。     

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.     

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.     

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.     

Inhibition of DNA methyltransferase inhibits DNA replication

Ectopic expression of DNA methyltransferase transforms vertebrate cells, and inhibition of DNA methyltransferase reverses the transformed phenotype by an unknown mechanism. We tested the hypothesis that the presence of an active DNA methyltransferase is required for DNA replication in human non-small cell lung carcinoma A549 cells. We show that the inhibition of DNA methyltransferase by two novel mechanisms negatively affects DNA synthesis and progression through the cell cycle. Competitive polymerase chain reaction of newly synthesized DNA shows decreased origin activity at three previously characterized origins of replication following DNA methyltransferase inhibition. We suggest that the requirement of an active DNA methyltransferase for the functioning of the replication machinery has evolved to coordinate DNA replication and inheritance of the DNA methylation pattern.     

真核生物的DNA甲基转移酶与DNA甲基化

真核生物的ＤＮＡ甲基化就是在ＤＮＡ的ＣｐＧ二核苷酸胞嘧啶的第 5位碳原子上加上甲基 ,催化这一过程的是ＤＮＡ甲基转移酶 (Ｄｎｍｔ)。ＤＮＡ的甲基化修饰参与基因表达调控、胚胎发育、细胞分化、基因组印迹、Ｘ染色体灭活和细胞记忆等诸多重要生物学过程[1,2 ] 。在不同组织或同一类型细胞的不同发育阶段 ,基因组ＤＮＡ上各ＣｐＧ位点甲基化状态的差异即构成基因组的ＤＮＡ甲基化谱。根据催化反应类型。可以将ＤＮＡ甲基转移酶分为三类 :第一类将腺嘌呤转化成Ｎ6 甲基腺嘌呤 ;第二类将胞嘧啶转化成Ｎ4 甲基胞嘧啶 ;第三类将胞嘧啶转化成…     

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.