期刊
CHEMBIOCHEM
卷 12, 期 3, 页码 431-438出版社
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201000644
关键词
base-modified DNA; DNA cleavage; DNA polymerases; nucleosides; restriction endonucleases
资金
- Academy of Sciences of the Czech Republic [Z40550506]
- Ministry of Education of the Czech Republic [LC512]
- Czech Science Foundation [203/09/0317]
- Gilead Sciences, Inc. (Foster City, CA, USA)
A series of six pyrimidine-modified dNTPs-5-ethynyl-, 5-phenyl-, and 5-(3-nitrophenyl)deoxycitidine and -deoxyuridine triphosphates-were prepared and incorporated by primer extension with Vent (exo-)polymerase to specific DNA sequences within or next to the recognition sequences of selected restriction endonucleases. The cleavage of these pyrimidine-modified DNA sequences by 13 restriction enzymes was then studied. Whereas the presence of any modified C within the target sequence completely prevented any restriction cleavage, most enzymes tolerated the presence of 5-ethynylU and two of them even the presence of 5-phenyl- and 5-(3-nitrophenyl)U. Modifications outside the recognition sequence were tolerated except in the case of phenyl derivatives with the PvuII enzyme. 5-EthynylC was used for protection of the recognition sequence from cleavage in the presence of the second unmodified copy of the same sequence that was cleaved.
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