In vitro mutagenesis by incorporation of N4-aminodeoxycytidine 5'-triphosphate.
We have investigated the possibility of introducing a new way to carry out in vitro mutagenesis. N4-Aminodeoxycytidine 5'-triphosphate was used in the Klenow enzyme-catalyzed chain elongation of a primer oligonucleotide hybridized to a lacZ alpha region of M13mp2 viral single strand DNA, and the possibility of inducing an efficient, randomly distributed point mutations into this particular genomic region was explored. On transfection of the resulting DNA into E. coli, mutant phages emerged at frequencies up to 1%. Analysis of the DNA sequences of the mutants has shown that single transitions, either A to G or G to A, were induced in a random fashion, thus providing data to show the possibility of using this method for production of mutant proteins having various single amino-acid changes in a defined domain.
Negishi
K
K
Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Matsumoto
K
K
Bessho
T
T
Tada
F
F
Hayatsu
H
H
D003090:Coliphages / Q000235:genetics; D004277:DNA, Single-Stranded / Q000187:drug effects / Q000235:genetics; D004279:DNA, Viral / Q000187:drug effects / Q000235:genetics*; D003845:Deoxycytosine Nucleotides / Q000378:metabolism / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D009153:Mutagens / Q000494:pharmacology*; D009154:Mutation
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D003845:Deoxycytosine Nucleotides; D009153:Mutagens; C046787:N(4)-amino-2'-deoxycytidine triphosphate