Modified DNA bearing 5(methoxycarbonylmethyl)-2'-deoxyuridine: preparation by PCR with thermophilic DNA polymerase and postsynthetic derivatization.
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.
Mehedi Masud
Mohammad
M
Department of Applied Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan.
Ozaki-Nakamura
Akiko
A
Kuwahara
Masayasu
M
Ozaki
Hiroaki
H
Sawai
Hiroaki
H
D000588:Amines / Q000737:chemistry; D004247:DNA / Q000096:biosynthesis* / Q000138:chemical synthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry; D006632:Histamine / Q000737:chemistry; D006868:Hydrolysis; D016133:Polymerase Chain Reaction; D019711:Pyrococcus / Q000201:enzymology; D013698:Templates, Genetic
D000588:Amines; D006632:Histamine; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D003857:Deoxyuridine