携带5-（甲氧基羰基甲基）-2'-脱氧尿苷的修饰DNA：通过嗜热DNA聚合酶PCR和合成后衍生化制备
Modified DNA bearing 5(methoxycarbonylmethyl)-2'-deoxyuridine: preparation by PCR with thermophilic DNA polymerase and postsynthetic derivatization.
作者信息
Mehedi Masud Mohammad, Ozaki-Nakamura Akiko, Kuwahara Masayasu, Ozaki Hiroaki, Sawai Hiroaki
机构信息
Department of Applied Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan.
出版信息
Chembiochem. 2003 Jul 7;4(7):584-8. doi: 10.1002/cbic.200200539.
A thymidine analogue bearing a methyl ester at the C5 position was accepted as a substrate by the thermophilic family B DNA polymerases, KOD Dash, Pwo, and Vent(exo-), to form the corresponding PCR product, but not by the thermophilic family A DNA polymerases, Taq, Tth, and T7 thermosequenase. Modified DNA containing this analogue was prepared by PCR on a large scale with KOD Dash DNA polymerase and 5(methoxycarbonylmethyl)-2'-deoxyuridine 5'-triphosphate as a substrate. The methyl ester of the modified DNA was further allowed to react with tris(2-aminoethyl)amine or histamine by an ester-amide exchange reaction to form the corresponding derivatized DNA bearing a tris(2-aminoethyl)amine or histamine moiety. Hydrolysis of the methyl ester of the modified DNA gave a functionalized DNA bearing an anionic carboxyl group. The derivatized DNA could act as a template for the PCR with KOD Dash DNA polymerase and the natural 2'-deoxythymidine 5'-triphosphate or the modified thymidine analogue as a substrate. The postsynthetic derivatization of the modified DNA may expand the variety of structurally modified DNA produced by PCR.
一种在C5位置带有甲酯的胸苷类似物被嗜热B族DNA聚合酶KOD Dash、Pwo和Vent（exo-）接受为底物，以形成相应的PCR产物，但嗜热A族DNA聚合酶Taq、Tth和T7热测序酶则不接受。以KOD Dash DNA聚合酶和5（甲氧基羰基甲基）-2'-脱氧尿苷5'-三磷酸为底物，通过PCR大规模制备含有这种类似物的修饰DNA。修饰DNA的甲酯进一步通过酯-酰胺交换反应与三（2-氨基乙基）胺或组胺反应，形成带有三（2-氨基乙基）胺或组胺部分的相应衍生化DNA。修饰DNA甲酯的水解产生带有阴离子羧基的功能化DNA。衍生化DNA可以作为KOD Dash DNA聚合酶进行PCR的模板，以天然的2'-脱氧胸苷5'-三磷酸或修饰的胸苷类似物作为底物。修饰DNA的合成后衍生化可能会扩大通过PCR产生的结构修饰DNA的种类。
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