Oligonucleotide-directed mutagenesis without phenotypic selection.
A DNA sequence can be specifically altered by synthesizing the desired sequence change within an oligonucleotide, and then converting this into a biologically active circular DNA strand by using the oligonucleotide to prime in vitro synthesis on a single-stranded circular template. This unit presents a protocol which uses a DNA template containing a small number of uracil residues in place of thymine. Use of the uracil-containing template allows rapid and efficient recovery of mutants; in principle this same template can be applied to most of the other mutagenesis protocols in use. The length of the oligonucleotide primer is highly variable and depends on the nature of the change being made.
Kunkel
T A
TA
National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.
D017104:Bacteriophage M13 / Q000235:genetics; D004247:DNA / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010641:Phenotype
D017931:DNA Primers; D004247:DNA