An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues

作者：Zaccolo M^*; Williams DM; Brown DM; Gherardi E

来源：Journal of Molecular Biology, 1996, 255(4): 589-603.

DOI：10.1006/jmbi.1996.0049

• pyrimidine analogue
• dP
• 8-oxodeoxyguanosine
• PCR
• random mutagenesis
• protein engineering

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 vitro with Tag polymerase and in the presence of the 5'-triphosphates of 6-(2-deoxy-beta-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido-[4,5-C] [1,2]oxazin-7-one(dP) and of 8-oxo-2'deoxyguanosine (8-oxodG). The newly synthesised triphosphate derivative of dP (dPTP) is an excellent substrate for Tag polymerase (K-m = 22 mu M versus K-m = 9.5 mu M for TTP); it is incorporated in place of TTP and, with a similar to 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 x 10(-2)), T-->C (4.3 x 10(-2)), G-->A (1.1 x 10(-2)) and C-->T (1.0 x 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 x 10(-2) and 1.2 x 10(-2) respectively. 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 x 10(-1)), (2) it allows control of the mutational load via the 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.

• 出版日期1996-2-2