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高盐沉淀CTAB法提取温室菊花基因组DNA 总被引:4,自引:0,他引:4
根据温室菊花植物组织富含多酚、多糖的具体特性,对CTAB法加以改进:在待沉淀液中加入1/2体积5 mol·L~NaCI.改进后的方法获得的DNA质量良好,电泳条带清晰,提取过程无明显的DNA降解,基本上排除了多酚物质的干扰.以提取的DNA为模板,用一对引物扩增菊花中18S基因,得到条带单一,大小与已知一致,说明获得的DNA可以进行PCR扩增,EcoR I 酶切基因组DNA图谱表明,提取的DNA能被限制性内切酶完全酶切,可以满足相关的分子生物学研究. 相似文献
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三种樟科植物的细胞总DNA提取 总被引:12,自引:0,他引:12
为了从富含次生代谢物的樟科植物肉桂、锡兰肉桂、阴香中获得高质量DNA ,研究和改进了CTAB法、高盐低pH法和尿素法。改进方法包括 :1)在裂解液中加入 2 % β -巯基乙醇和 5 %PVP ,以防止氧化褐变的发生 ;2 )在酚 :氯仿抽提前加入 1 5mol L醋酸铵冰浴处理 ,能降低DNA的黏性。所得DNA的质量和产量经电泳、紫外吸收A2 60 A2 80 、PCR扩增和限制性内切酶酶切检测 ,结果表明改进法提取的DNA质量要比常规法的好。其中改进的CTAB法获得的DNA纯度最高 ,能用于PCR扩增和限制性内切酶酶切 ,是提取这 3种樟科植物总DNA的最佳方法。 相似文献
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降香黄檀基因组DNA的提取方法研究 总被引:2,自引:0,他引:2
目的:建立适合降香黄檀基因组DNA的提取方法。方法:采用常规SDS法、常规CTAB法和改良CTAB法等3种方法提取降香黄檀叶片基因组DNA,经电泳、吸光度、酶切检测比较提取结果;对采用改良CTAB法提取的基因组DNA进行ISSR-PCR检测。结果:改良CTAB法通过增加洗涤样品步骤,有效去除了多糖和多酚类物质,提取的DNA质量好,无降解现象,无蛋白质、盐离子及RNA污染。结论:改良CTAB法是一种高效的提取方法,使用该方法所得DNA的质量完全能够满足相应的分子操作需要。 相似文献
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梨不同DNA提取方法的效果研究 总被引:28,自引:0,他引:28
以7个梨品种为实验材料,比较分析了SDS法、CTAB法、SDSCTAB法、改良的CTAB法、高盐低pH值法、分步离心法对梨总DNA提取的效果。结果表明:利用以上6种方法提取的梨总DNA在纯度和量上有很大的差别。所得到的平均DNA量从大到小依次为:分步离心法、SDS法、SDSCTAB法、改良的CTAB法、CTAB法、高盐低pH值法。DNA提取纯度依次为分步离心法、SDSCTAB法、改良的CTAB法、高盐低pH值法、CTAB法、SDS法。RAPD和自交不亲和基因(S基因)特异性引物扩增实验结果都比较理想,但分步离心法和SDSCTAB法提取的DNA双酶切效果较好。分步离心法提取的梨总DNA更适用于后续的分子生物学实验操作。 相似文献
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本研究采用改良SDS法、常规CTAB法和改良CTAB法(1.5×CTAB,2×CTAB,3×CTAB)提取大青杨基因组DNA,并用紫外光普分析、凝胶电泳、限制性内切酶消化和RAPD方法进行鉴定.结果表明:5种方法中,改良SDS法DNA提取率最高,但CTAB法比改良SDS法提取获得的DNA纯度高,OD260/OD280为1.73~1.81.与常规CTAB法比较,改良SDS法和改良CTAB法能有效去除蛋白、多糖、酚类及次生代谢物质.综合分析确定改良2×CTAB法为大青杨基因组DNA的最佳提取方法. 相似文献
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Capping DNA with DNA 总被引:13,自引:0,他引:13
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. 相似文献
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Eukaryotic DNA polymerases in DNA replication and DNA repair 总被引:16,自引:0,他引:16
Peter M. J. Burgers 《Chromosoma》1998,107(4):218-227
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 相似文献
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Hans V. Westerhoff Mary H. O’Dea Anthony Maxwell Martin Gellert 《Cell biochemistry and biophysics》1988,12(1):157-181
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. 相似文献
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DNA topoisomerases and DNA repair 总被引:5,自引:0,他引:5
C S Downes R T Johnson 《BioEssays : news and reviews in molecular, cellular and developmental biology》1988,8(6):179-184
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. 相似文献
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D M Lilley 《Biochemical Society transactions》1986,14(2):211-213
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《Cell cycle (Georgetown, Tex.)》2013,12(9):1339-1340
Comment on: Witz G, et al. Proc Natl Acad Sci USA 2011; 108:3608-11. 相似文献
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DNA杂交与DNA指纹技术 总被引:1,自引:0,他引:1
Southern印迹杂交和DNA指纹技术在分子生物学研究以及疾病的诊断、亲缘关系鉴定、犯罪分子确认等过程中发挥了重要作用。回顾了2种技术的发明、发展历程和在生命科学研究中的作用,并探讨了可能的发展方向,从中可以从一个侧面了解分子生物学的发展历程和体会科学家的智慧在科学技术发展中所起的重要作用。 相似文献
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DNA supercoiling inhibits DNA knotting 总被引:1,自引:1,他引:0
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. 相似文献
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DNA methylation on cytosine is an epigenetic modification and is essential for gene regulation and genome stability in vertebrates. Traditionally DNA methylation was considered as the most stable of all heritable epigenetic marks. However, it has become clear that DNA methylation is reversible by enzymatic “active” DNA demethylation, with examples in plant cells, animal development and immune cells. It emerges that “pruning” of methylated cytosines by active DNA demethylation is an important determinant for the DNA methylation signature of a cell. Work in plants and animals shows that demethylation occurs by base excision and nucleotide excision repair. Far from merely protecting genomic integrity from environmental insult, DNA repair is therefore at the heart of an epigenetic activation process. 相似文献