Modified DNA Bearing 5(Methoxycarbonylmethyl)-2′-deoxyuridine: Preparation by PCR with Thermophilic DNA Polymerase and Postsynthetic Derivatization

Department of Applied Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan.
ChemBioChem (Impact Factor: 3.09). 07/2003; 4(7):584-8. DOI: 10.1002/cbic.200200539
Source: PubMed


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.

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    • "The presence of a charge near the nucleobase decreased the yield of the PCR products. Our previous study showed that the analog TME, which has no charge on the modified group, acts as a good substrate for PCR catalyzed by KOD Dash, whereas 5-(2-hydroxy-2-oxoethyl)-dUTP, which has a negative charge on the modified group, does not (30). Therefore, the amount of PCR products is decreased by either a negative or positive charge on the modified group. "
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