Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerases.
Effects of 2-chloro-2'-deoxyadenosine 5'-triphosphate on DNA synthesis in vitro by purified bacterial and viral DNA polymerases.
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2-氯-2-脱氧腺苷 5-三磷酸对纯化的细菌和病毒 DNA 聚合酶体外 DNA 合成的影响。
DOI：
10.1021/bi00216a032
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Blakley,RL
中科院分区：
文献类型：
--
作者：
Hentosh,P;McCastlain,JC;Blakley,RL
材料与方法材料。AMV RT、噬菌体T4 DNA聚合酶、双脱氧核苷酸测序试剂、甲酰胺停止液(95%甲酰胺、20 mM EDTA、0.05%溴酚蓝、0.05%二甲苯酚FF)和序列酶购自美国生物化学公司。大肠杆菌DNA聚合酶I(Klenow片段)、M13mpl8(+)链DNA、引物1[与M13mpl8(+)strand 6327-6311位互补的17碱基脱氧核苷酸(5‘-GTTTCCCAGCACACGAC-3’)]和引物2[5‘-GTAAAACGGCAGGT-3’，其退火位位于M13mpl8的6307-6291位。分子生物学级别的dNTPs，引物3[5‘-GTCATAGCTGTTTCCTG-3’，与M13mpl8的6221-6205碱基互补]，丙烯酰胺来自Bio-rad，柯达XAR和杜邦Cronex胶片来自Med Cor X-ray Systems。Nensorb柱、[a-32PJdATP]和[y-32P]ATP来自新英格兰核。T4多核苷酸激酶来自贝塞斯达研究实验室。圣犹大儿童研究医院分子资源中心也在一台应用生物系统380B DNA合成仪上生产了M13MP 18特异性引物。CldAdo按所述方法合成(Huang等人，1981)，三磷酸衍生物由标准工艺合成。用高效液相分析法鉴定CldATP的纯度。没有检测到污染正常的dNTPs，并且很少或没有检测到单磷酸和二磷酸形式(<；1%)。HIV-1 RT是马里兰州贝塞斯达国家癌症研究所的Hiroaki Mitsuya博士的礼物。5‘-末端标记的DNA体外合成反应。如报道的那样，M13mpl8特异性引物2和3被T4多核苷酸激酶和[y-32P]ATP通过正向激酶反应5‘末端标记(Hentosh等人，1990年)。标记的引物与M13mpl8(+)链DNA退火，并在三个正常dNTP存在的情况下用于引物延伸反应，并将CldATP或dATP作为第四个核苷酸(Hentosh等人，1990年)。每个聚合酶的最终引物延伸反应缓冲液在6.5PU中包含以下内容。对于AMV RT，50 mM Tris-HCl(pH 8.3)，6 mM MgCl2，40 mM KCl，1 mM DTT，和2-4个单位的AMV RT(48245个单位/毫克)；对于Klenow片段，10 mM Tris-HCl(pH 7.5)，5 mm MgCl2，2.5 mM DTT，和0.05-0.1个单位的Klenow片段(15000个单位/mg)；对于序列酶，10 mM Tris-HCl(pH 7.5)，5 mm MgCl2，35 mM NaC l，3.5 mm DTT，和0.013个单位的序列酶(30000个单位/mg)；T4DNA聚合酶，67 mM Tris-HCl(pH 8.8)。6.7mMMgCl2、16.6 mM(NH4)2S04、10 mM 2-巯基乙醇和0.6单位T4DNA聚合酶(33 300单位/mg)；对于HIV-1 RT，50 mM Tris-HCl(PH 8)，100 mM NaC l，8 mM DTT和0.4 pg HIV-1 RT。每种酶的单位定义如制造商所述：对于Sequenase，Klenow片段，和T4，1
Materials and Methods Materials. AMV RT, phage T4 DNA polymerase, di-deoxynucleotide sequencing reagents, formamide stop solution (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% Xylene cyanol FF), and Sequenase were purchased from United States Biochemical Corp. Escherichia coli DNA polymerase I (Klenow fragment), M13mpl8 (+) strand DNA, primer1[a 17-base deoxynucleotide(5'-GTTTTCCCAGTCACGAC-3') complementary to positions 6327-6311 of M13mpl8 (+) strand], and primer 2 [5'-GTAAAACGACGGCCAGT-3', which anneals to positions 6307-6291 of M13mpl8] were obtained from New England Biolabs. Molecular biology grade dNTPs, primer 3 [5'-GTCATAGCTGTTTCCTG-3', complementary to bases 6221-6205 of M13mpl8], and acrylamide were from Bio-rad, and Kodak XAR and Dupont Cronex film were from Med Cor X-ray Systems. Nensorb columns,[a-32PJdATP, and [y-32P] ATP were from New England Nuclear. T4 polynucleotide kinase was from Bethesda Research Laboratories. M13mp 18-specific primers were also produced in the Molecular Resource Center at St. Jude Children’s Research Hospital on an Applied Biosystems 380B DNA synthesizer. CldAdo was synthesized as described (Huang et al., 1981), and triphosphate derivatives were produced fromit by standard techniques. The purity of CldATP was assessed by analytical HPLC. No contaminating normal dNTPs were detected, and very little or no mono-and diphosphate forms were detected (< 1%). HIV-1 RT was the gift of Dr. Hiroaki Mitsuya, National Cancer Institute, Bethesda, MD. In Vitro DNA Synthesis Reactions Using 5'-End-Labeled Primers. M13mpl8-specific primers 2 and 3 were 5'-endlabeled with T4 polynucleotidekinase and [y-32P] ATP by the forward kinase reaction as reported (Hentosh et al., 1990). Labeled primer was annealed to M13mpl8 (+) strand DNA and used in primer extension reactions in the presence of three normal dNTPs and either CldATP or dATP as the fourth nucleotide as described (Hentosh et al., 1990). Final primer extension reaction buffersfor each polymerase contained the following in 6.5 pU. for AMV RT, 50 mM Tris-HCl (pH 8.3), 6 mM MgCl2, 40 mM KC1, 1 mM DTT, and 2-4 units of AMV RT (48 245 units/mg); forKlenow fragment, 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2.5 mM DTT, and 0.05-0.1 unit of Klenow fragment (15 000 units/mg); for Sequenase, 10 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 35 mM NaCl, 3.5 mM DTT, and 0.013 unit of Sequenase (30000 units/mg); forT4 DNA polymerase, 67 mM Tris-HCl (pH 8.8). 6.7 mMMgCl2, 16.6 mM (NH4) 2S04, 10 mM 2-mercaptoethanol, and 0.6 unit ofT4 DNA polymerase (33 300 units/mg); for HIV-1 RT, 50 mM Tris-HCl (pH 8.), 100 mM NaCl, 8 mM DTT, and 0.4 pg of HIV-1 RT. The unit definitions for each enzyme were as stated by the manufacturer: for Sequenase, Klenow fragment, and T4, 1
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影响因子：
11.2
作者：
J. Griffig;R. Koob;Raymond L. Blakley
通讯作者：
J. Griffig;R. Koob;Raymond L. Blakley
影响因子：
2.9
作者：
LADUCA, RJ;FAY, PJ;BAMBARA, RA
通讯作者：
BAMBARA, RA
DOI：
10.1073/pnas.84.14.4767
发表时间：
1987-07-01
影响因子：
11.1
作者：
TABOR, S;RICHARDSON, CC
通讯作者：
RICHARDSON, CC
影响因子：
11.4
作者：
BERNAD, A;ZABALLOS, A;BLANCO, L
通讯作者：
BLANCO, L
人类免疫缺陷病毒 I 型逆转录酶复制天然 DNA 的保真度。
影响因子：
14.9
作者：
J. Weber;Frank Grosse
通讯作者：
Frank Grosse