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

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.