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小鼠DNA的限制性片段长度多态性分析 总被引:2,自引:0,他引:2
用人类肌红蛋白基因内含子中33bp的小卫星DNA重复序列为探针,经pBR322的EcoR I正向测序引物和α-32P-dCTP在Taq DNA聚合酶作用下做合成标记,与6种小鼠的DNA限制性片段杂交。结果显示,每个品系平均出现可分辨带纹在13条以上,不同品系小鼠间的杂交带纹表现出高度的多态性。多态性带纹主要分布在6 ̄23kb区段;同一近交系的不同个体间的带纹特点及分布完全一致;CS7与AKR及二者 相似文献
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人体小卫星DNA探针的制备 总被引:3,自引:2,他引:1
根据人体小卫星DNA核心顺序,化学合成长23碱基寡核苷酸探针,筛选人体基因组文库,旨在获得能用作遗传分析探针的小卫星顺序。结果得到15个含小卫星的阳性重组子。随机取其一(C_(35.9))作探针,试做群体分析。所有个体均可检出多条杂交带。其中某些带具有多态性。在一定检测条件下,检出的DNA图谱在有限的个体内具有个体特异性。结果表明筛选文库得到的小卫星顺序可用于小卫星多态性的检测。其它小卫星探针的筛选和应用性研究正在进行。 相似文献
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菜豆基因组微卫星DNA的分离与鉴定 总被引:2,自引:0,他引:2
从耐高温型菜豆(Phaseolus vulgaris L.)品种“Haibushi”基因组分离微卫星DNA,可以提供与耐热性QTL(quantitative trait locus)连锁的多态遗传标记。为此,利用lambda ZAPⅡ载体构建了一个包含400-800bp插入片段的菜豆基因组文库。利用地高辛末端标记寡聚核苷酸(GA)10和(CA)10作为探针筛选该文库,并对部分阳性克隆进行测序分析, 相似文献
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由小麦*玉米获得的普通小麦加倍单倍体后代的RFLP变异 总被引:1,自引:1,他引:0
用小麦(TriticumaestivumL.)的rDNA克隆pTa71和与小麦基因组有部分同源性的玉米(ZeamaysL.)DNA克隆作探针,对由小麦*玉米获得的普通小麦加倍单倍体后工群体进行RFLP分析。 相似文献
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编码天麻抗真菌蛋白cDNA的分子克隆 总被引:9,自引:0,他引:9
用重组DNA 技术研究了编码天麻抗真菌蛋白(GAFP)的基因,从天麻(Gastrodia elataBl.)块茎中提取Poly(A) m RNA 后合成cDNA,构建成表达型cDNA 文库,用纯化的蛋白质探针通过免疫筛选找出对应的cDNA 克隆。在进一步证明所选用的cDNA 克隆含有重组的λ-phage DNA 后,提取和纯化含有插入片段重组子的DNA,用Eco RI酶切分析可见插入片段。已分离出编码天麻抗真菌蛋白的基因 相似文献
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单纯疱疹病毒(HSV)Ⅰ型及Ⅱ型之间有很多共同抗原,能引起血清学交叉反应,鉴别诊断比较困难。本实验利用重组DNA技术,将部分HSV-2DNA的PstI片段克隆到载体质粒PSK中,并筛选出两个重组质粒(P和P)只与HSV-2反应,与HSV-1不反应,这两个重组质粒中所含的HSV-2DNA片段大小分别是3.1和4.3kb,另外,还筛选了一个重组质粒(PHSV2-1,含5.8kbHSV-2DNA片段)与HSV-1和HSV-2均反应。将4.3kb的片段用光生物素标记后作为探针检测了159份人阴道拭子,其中23份样品呈阳性反应,其余均为阴性,从23份阳性样品中挑选12价涂片用间接荧光抗体法检测也都呈阳性反应,随机挑选的几份杂交反应阴性样品在间接荧光试验中也是阴性。本实验制备的HSV通用及HSV-2型特异性探针将比常规的血清学方法诊断和鉴别HSV-1和HSV-2感染更为可靠。 相似文献
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以p53cDNA为探针,用Southern印迹法对人胃癌细胞系BGC823进行了检测,发现该细胞中p53基因存在异常,将可在真核细胞表达的重组野生型P53质粒pC53-SN3和突变型p53质粒pC53-SCX3,用指质体介导法,分别导入BCG823细胞,获得了较长时间受G418的多个阳性克隆。Southern抑迹法证实阳性克隆细胞中有外源性p53基因存在。 相似文献
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为了克隆到全长的人促红细胞生成素基因,从而在真核系统中及转基因动物乳腺中进行表达,从一个中国人的血细胞中提取基因组DNA,构建了不完全基因组噬菌体文库,文库效价为5×104.这种文库的构建方法是用BamHⅠ对基因组DNA进行完全酶切,回收10.5kb左右的酶切片段,插入到λ-噬菌体EMBL3载体中.筛选文库所使用的探针是用PCR方法从基因组DNA模板中扩增的556bp的EPO基因片段.从该文库中筛选到了一个含有完整EPO基因的插入片段长度为12.5kb的阳性克隆.经全序列分析证实,所克隆的EPO基因编码正确,但在5′-侧翼及第一内含子处与国外发表的序列相比较,发现两处核苷酸差异,这两个核苷酸的差异改变了5′-调控区的两个小的开放阅读框——ORF1和ORF3,其在基因表达调探方面的作用值得进一步探讨. 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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D M Lilley 《Biochemical Society transactions》1986,14(2):211-213
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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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《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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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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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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Human placental DNA methyltransferase: DNA substrate and DNA binding specificity 总被引:9,自引:5,他引:4 下载免费PDF全文
We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally has virtually all of its cytosine residues replaced by 5-methylcytosine (m5C). Micrococcus luteus DNA was just as good a substrate if it was first similarly nick translated with m5dCTP instead of dCTP in the polymerization mixture. At different stages in purification and under various conditions (including in the presence or absence of high mobility group proteins), the methylation of m5C-deficient DNA and that of hemimethylated DNA were compared. Although hemimethylated , m5C-rich DNAs were much better substrates than were m5C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme. In contrast, from the same placental extract, a DNA-binding protein of unknown function was isolated which binds to m5C-rich DNA in preference to the analogous m5C-poor DNA. 相似文献