Mark Mcdougall

ANALOGS OF GUANINE NUCLEOSIDE TRIPHOSPHATES FOR SEQUENCING APPLICATIONS

We have synthesized more than 30 different deoxyribonucleosides and triphosphates with modifications either in the base or the phosphate moiety as analogs of 2′-dGTP for DNA sequencing applications. All the modified nucleoside triphosphates were tested as substrates for DNA polymerases, including Sequenase™ T7 DNA polymerase or Thermo Sequenase™ DNA polymerase. Two of the analogs, 7-ethyl-7-deaza-dGTP and 7-hydroxymethyl-7-deaza- dGTP meet our requirements as better sequencing reagents.

Synthesis and enzymatic incorporation of a novel, bicyclic pyrimidine nucleoside: a thymidine mimic

Abstract Nucleophilic ring-opening and rearrangement reaction of a furanopyrimidine nucleoside with anhydrous hydrazine provided a novel, 6,6-bicyclic pyrimidopyridazin-7-one nucleoside (dH, 4 ), whose structure was confirmed by X-ray crystallography. This novel nucleoside was converted to its 5′-triphosphate (dHTP) for studies with DNA polymerases and incorporated into a template by using standard phosphoramidite chemistry. In the template, dH directed the incorporation of dATP and to a lesser extent dGTP into the transcript and dHTP was efficiently incorporated at the 3′-end of a primer opposite dA using both exonuclease free Klenow fragment (KF exo-) and Taq DNA polymerases and extended with natural dNTPs.

Random Mutagenesis Using 2-Amino-9-(2-Deoxy-β-D-Ribofuranosyl)Purine-5′-Triphosphate and the Polymerase Chain Reaction

Abstract Base analogues offer an attractive method for mutagenising DNA in combination with the polymerase chain reaction (PCR). We have synthesised the 5′-triphosphate-2′-deoxyribosyl derivative of 2-aminopurine (dAPTP), one of the first base analogues to be used for mutagenesis, and examined its utility in PCRs. An E. coli amber suppressor gene, supF, was used as a template for mutagenesis. The analogue induced exclusively transition mutations, but at a low frequency, consistent with its weak mutagenicity in vivo.