Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Gilead Sciences & IOCB Research Center, Flemingovo náměstí 2, Prague 6, 16610, Czech Republic.
Chembiochem. 2011 Feb 11;12(3):431-8. doi: 10.1002/cbic.201000644. Epub 2011 Jan 14.
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.
一系列六种嘧啶修饰的 dNTPs——5-乙炔基、5-苯基和 5-(3-硝基苯基)脱氧胞苷和脱氧尿苷三磷酸——被制备并通过引物延伸与 Vent(exo-)聚合酶掺入到选定的限制内切酶的识别序列内或附近的特定 DNA 序列中。然后研究了 13 种限制酶对这些嘧啶修饰的 DNA 序列的切割。尽管靶序列中任何修饰的 C 的存在完全阻止了任何限制切割，但大多数酶容忍 5-乙炔基 U 的存在，其中两种酶甚至容忍 5-苯基和 5-(3-硝基苯基)U 的存在。修饰物在识别序列之外是可以容忍的，除了 PvuII 酶与苯衍生物的情况。5-乙炔基 C 用于保护识别序列免受同一序列的第二个未修饰副本的切割。
