deoxyguanosine-triphosphate and dimethyl-sulfate

The 5'-triphosphate of the boronated nucleoside analog N7-cyanoborane-2'-deoxyguanosine (7bdGTP) was synthesized, and a series of experiments was initiated to assess the potential of the compound to serve as a substrate for DNA polymerases. We show here that 7bdGTP can be incorporated into DNA by Sequenase. The resulting hemiboronated extension products are resistant to cleavage by treatment with either DMS and heat or a number of restriction enzymes. Further, in the polymerase chain reaction, 7bdGTP can be utilized as a substrate for Taq polymerase. Finally, by kinetic analysis, we have found that 7bdGTP is a more efficient substrate for exonuclease-free Klenow than normal dGTP. Thus, the introduction of a cyanoborane moiety to the N7 position of dGTP results in a nucleotide that is accepted in lieu of normal dGTP by a number of DNA polymerases.

Topics: Base Sequence; Boron Compounds; Deoxyguanine Nucleotides; DNA Primers; DNA Restriction Enzymes; DNA-Directed DNA Polymerase; In Vitro Techniques; Kinetics; Molecular Sequence Data; Polymerase Chain Reaction; Substrate Specificity; Sulfuric Acid Esters; Taq Polymerase