Synthesis of N7-ethyldeoxyguanosine 5'-triphosphate and placement of N7-ethylguanine in a specific site in a synthetic oligodeoxyribonucleotide.
Synthesis of N7-ethyldeoxyguanosine 5'-triphosphate and placement of N7-ethylguanine in a specific site in a synthetic oligodeoxyribonucleotide.
复制标题
N7-乙基脱氧鸟苷 5-三磷酸的合成以及将 N7-乙基鸟嘌呤放置在合成寡脱氧核糖核苷酸的特定位点。
DOI：
10.1016/0003-2697(89)90200-5
复制
发表时间：
1989
影响因子：
2.9
通讯作者：
Ivarie,R
中科院分区：
文献类型：
--
作者：
Farrance,IK;Ivarie,R
N7-乙基脱氧鸟苷 5'-三磷酸 (N7-EtdGRP) 是通过硫酸二乙酯直接乙基化 dGTP 合成的，并通过纤维素平板上的 TLC 纯化，收率约为 5%。N7-EtdGTP 通过其在 pH 1、7.4 和 13 下的紫外光谱、吸光度最大值和最小值以及糖苷键对酸和酸的不稳定性来鉴定。热诱导裂解。在 pH 7.4 时，糖苷键自发裂解，半衰期 >48 小时。使用末端脱氧核苷酸转移酶和大肠杆菌 DNA 聚合酶 I 的 Klenow 片段的 3' 至 5' 核酸外切酶和 5' 至 3' 聚合酶开发了一种将 N7-乙基鸟嘌呤置于 DNA 中特定位点的酶促方法。该方法应该很容易适应其他修饰的碱基，只要修饰不发生在碱基配对位点（例如， 5-甲基胞嘧啶、N6-甲基腺嘌呤等）。
N7-Ethyldeoxyguanosine 5′-triphosphate (N7-EtdGRP) was synthesized by direct ethylation of dGTP with diethyl sulfate and purified by TLC on cellulose plates at ∼5% yield.N7-EtdGTP was identified by its uv spectra at pH 1, 7.4, and 13, by its absorbance maxima and minima, and by the lability of the glycosidic bond to acid- and heat-induced cleavage. At pH 7.4, spontaneous cleavage of the glycosidic bond proceeded with a half-life of >48 h. An enzymatic method for placing anN7-ethylguanine in a specific site in DNA was developed using terminal deoxynucleotidyltransferase and the 3′ to 5′ exonuclease and 5′ to 3′ polymerase of the Klenow fragment ofEscherichia coli DNA polymerase I. The method should be readily adaptable to other modified bases as long as the modification does not occur at a base-pairing site (e.g., 5-methylcytosine,N6-methyladenine, and others).