Publication Date:
1985-07-19
Description:
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.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Myers, R M -- Lerman, L S -- Maniatis, T -- New York, N.Y. -- Science. 1985 Jul 19;229(4710):242-7.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/2990046" target="_blank"〉PubMed〈/a〉
Keywords:
Animals
;
Base Sequence
;
*Cloning, Molecular
;
DNA Restriction Enzymes
;
*DNA, Recombinant
;
DNA, Single-Stranded/genetics
;
Electrophoresis, Polyacrylamide Gel
;
Escherichia coli/genetics
;
Genetic Vectors
;
Mice
;
*Mutation
;
Nucleic Acid Denaturation
;
Plasmids
;
Templates, Genetic
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
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