A general method for saturation mutagenesis of cloned DNA fragments.
A new procedure for generating and isolating random single-base substitutions in cloned DNA fragments is presented. The mutations are generated by treatment of single-stranded DNA with various chemicals, followed by the synthesis of the complementary strand with reverse transcriptase. Misincorporation frequently occurs when the enzyme encounters a damaged base in the mutagenized template DNA. The resulting duplex DNA fragments containing random single-base substitutions are cloned, amplified as a population, and isolated from wild-type DNA by preparative denaturing gradient gel electrophoresis. The physical separation of mutant DNA fragments makes it possible to isolate and characterize large numbers of site-directed single-base substitutions in the absence of a phenotypic selection. This procedure should be generally applicable to the fine-structure genetic analysis of regulatory and protein-coding sequences.
Myers
R M
RM
Lerman
L S
LS
Maniatis
T
T
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant; D004277:DNA, Single-Stranded / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D051379:Mice; D009154:Mutation; D009691:Nucleic Acid Denaturation; D010957:Plasmids; D013698:Templates, Genetic
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D004262:DNA Restriction Enzymes