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Science 19 July 1985:
Vol. 229. no. 4710, pp. 242 - 247
DOI: 10.1126/science.2990046
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Articles

Science, Vol 229, Issue 4710, 242-247
Copyright © 1985 by American Association for the Advancement of Science

articles

A general method for saturation mutagenesis of cloned DNA fragments

RM Myers, LS Lerman, and T Maniatis

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.


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