Induction of transition and transversion mutations during random mutagenesis PCR the addition of 2-hydroxy-dATP

Abstract

A 2-substituted purine nucleotide analog, 2-hydroxy-2′-deoxyadenosine 5′-triphosphate (2-OH-dATP), was added to a PCR mixture, to examine its mutagenic potential. The 2-OH-dATP enhanced the total mutation frequency. Interestingly, 2-OH-dATP induced both transition and transversion mutations, including A:T→G:C, A:T→C:G and G:C→T:A mutations. In contrast, other 2-substituted purine nucleotide analogs, 2-aminopurine-2′- deoxyriboside 5′-triphosphate and 2-amino-2′-deoxyadenosine 5′-triphosphate, did not affect the total mutation frequency. These results suggest that 2-OH-dATP is useful in random PCR mutagenesis for the in vitro evolution of nucleic acids and proteins, and for analyses of residues in these biomolecules. © 2004 Pharmaceutical Society of Japan.