An Approach to Random Mutagenesis of DNA Using Mixtures of Triphosphate Derivatives of Nucleoside Analogues

Abstract

We describe a new method for random mutagenesis of DNA based on the use of a mixture of triphosphates of nucleoside analogues. The method relies on DNA amplification in vitrowith Taqpolymerase and in the presence of the 5′-triphosphates of 6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihy dro-8H-pyrimido-[4,5-C] [1,2]oxazin-7-one(dP) and of 8-oxo-2′deoxyguano- sine (8-oxodG). The newly synthesised triphosphate derivative of dP (dPTP) is an excellent substrate for Taqpolymerase ( K m=22 μM versus K m=9.5 μM for TTP); it is incorporated in place of TTP and, with a ∼fourfold lower efficiency, in place of dCTP. After 30 cycles of DNA amplification, equimolar mixtures of the four normal dNTPs and dPTP yield the following frequencies of the four transition mutations: A→G (4.4 × 10 −2), T→C (4.3 × 10 −2), G→A (1.1 × 10 −2) and C→T (1.0 × 10 −2). The triphosphate derivative of 8-oxodG (8-oxodGTP) is incorporated opposite template adenine and yields two transition mutations (A→C and T→G) at frequencies of 0.8 × 10 −2and 1.2 × 10 −2respectively. Reaction mixtures containing dPTP and 8-oxodGTP results in both dP and 8-oxodG-induced mutations and an extensive array of codon changes in the absence of insertions and deletions. The method described differs from previous mutagenesis procedures in three respects: (1) it enables very high frequencies of base substitutions (up to 1.9 × 10 −1), (2) it allows control of the mutational load viathe number of DNA amplification cycles and (3) it yields both transition and transversion mutations. The procedure may find application in the generation of libraries of DNA and protein mutants from which species with improved or novel activities may be selected.