An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues.
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 Taq 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 Taq polymerase (Km = 22 microM versus Km = 9.5 microM for TTP); it is incorporated in place of TTP and, with a approximately 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.
Zaccolo
M
M
ICRF Cell Interactions Laboratory, MRC Centre, Cambridge, UK.
Williams
D M
DM
Brown
D M
DM
Gherardi
E
E
D000595:Amino Acid Sequence; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000737:chemistry / Q000494:pharmacology*; D003854:Deoxyribonucleotides / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016296:Mutagenesis; D009153:Mutagens / Q000737:chemistry / Q000494:pharmacology*; D016133:Polymerase Chain Reaction / Q000379:methods; D019914:Taq Polymerase; D013698:Templates, Genetic
C097986:6-(2-deoxyribofuranosyl)-3,4-dihydro-8H-pyrimido(4,5-c)(1,2)oxazin-7-one 5'-triphosphate; D004279:DNA, Viral; D003848:Deoxyguanine Nucleotides; D003854:Deoxyribonucleotides; D009153:Mutagens; C078206:8-oxodeoxyguanosine triphosphate; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase