Induction of various mutations during PCRs with manganese and 8-hydroxy-dGTP

Abstract

To induce various mutations randomly, PCRs with Mn2+ and with a mutagenic deoxyribonucleotide, 8-hydroxy-dGTP (8-OH-dGTP), were performed. Mutations were induced by deoxyribonucleotide imbalance plus 500 μM Mn 2+ in the Mn2+-PCR, and the amplified DNA was inserted into a plasmid. The plasmid library obtained from the transformed bacterial cells was then used as the template in the next PCR, which was done with 50 or 100 μM 8-OH-dGTP. Four kinds of mutations, A:T→G:C and G:C→A:T transitions and A:T→T:A and A:T→C:G transversions, occurred with similar frequencies. These results suggest that this strategy will be useful in random PCR mutagenesis for the in vitro evolution of nucleic acids and proteins, and for analyses of residues in these biomolecules. © 2007 Pharmaceutical Society of Japan.