NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.
NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides.
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DOI：
10.1073/pnas.92.8.3091
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发表时间：
1995-04
影响因子：
11.1
通讯作者：
D. Zimmer;D. Crothers
中科院分区：
文献类型：
--
作者：
D. Zimmer;D. Crothers
描述了用于 NMR 研究的毫克量的统一 13C15N 标记 DNA 寡核苷酸的酶促合成程序。从在 13C 和 15N 营养源上生长的微生物获得的脱氧核苷酸被酶促磷酸化为 dNTP，并且使用 DNA 聚合酶 I 的 Klenow 片段的 3'-5' 核酸外切酶缺陷突变体和设计用于有效分离标记的寡核苷酸模板引物将 dNTP 掺入寡核苷酸中。 来自未标记模板的产物 DNA。标记策略已用于统一标记 DNA 双链体 dGGCAAAACGG.dCCGTTTTGCC 中的一条或另一条寡核苷酸链，以便于通过 NMR 进行分配和结构确定。介绍了 15N 和 13C 异核 NMR 实验在同位素标记 DNA 中的应用。
A procedure for the enzymatic synthesis of uniformly 13C15N-labeled DNA oligonucleotides in milligram quantities for NMR studies is described. Deoxynucleotides obtained from microorganisms grown on 13C and 15N nutrient sources are enzymatically phosphorylated to dNTPs, and the dNTPs are incorporated into oligonucleotides using a 3'-5' exonuclease-deficient mutant of Klenow fragment of DNA polymerase I and an oligonucleotide template primer designed for efficient separation of labeled product DNA from unlabeled template. The labeling strategy has been used to uniformly label one or the other oligonucleotide strand in the DNA duplex dGGCAAAACGG.dCCGTTTTGCC in order to facilitate assignment and structure determination by NMR. Application of 15N and 13C heteronuclear NMR experiments to isotopically labeled DNA is presented.