Synthesis of N7-ethyldeoxyguanosine 5'-triphosphate and placement of N7-ethylguanine in a specific site in a synthetic oligodeoxyribonucleotide.
N7-Ethyldeoxyguanosine 5'-triphosphate (N7-Etd-GTP) was synthesized by direct ethylation of dGTP with diethyl sulfate and purified by TLC on cellulose plates at approximately 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 greater than 48 h. An enzymatic method for placing an N7-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 of Escherichia 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).
Farrance
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Department of Genetics, University of Georgia, Athens 30602.
Ivarie
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D003848:Deoxyguanine Nucleotides / Q000138:chemical synthesis*; D004926:Escherichia coli / Q000201:enzymology; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis*; D010761:Phosphorus Radioisotopes; D010880:Piperidines / Q000032:analysis; D013056:Spectrophotometry, Ultraviolet
D003848:Deoxyguanine Nucleotides; D009838:Oligodeoxyribonucleotides; D010761:Phosphorus Radioisotopes; D010880:Piperidines; C060538:N(7)-ethyldeoxyguanosine 5'-triphosphate; C022007:7-ethylguanine; D006147:Guanine