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NMR of Enzymatically Synthesized Uniformly 13C15N-Labeled DNA Oligonucleotides
Daniel P. Zimmer and Donald M. Crothers
Proceedings of the National Academy of Sciences of the United States of America
Vol. 92, No. 8 (Apr. 11, 1995), pp. 3091-3095
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/2367010
Page Count: 5
You can always find the topics here!Topics: DNA, Oligonucleotides, Product labeling, Chemical equilibrium, Nucleotides, Nucleic acids, Protons, Hydrolysis, Duplexes, RNA
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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.
Proceedings of the National Academy of Sciences of the United States of America © 1995 National Academy of Sciences