Simple approach for oligonucleotide-directed mutagenesis of any double-stranded circular DNA.
A new site-directed method for introducing mutations into any region of plasmid vector close to the unique restriction site is described. It is based on the use of 5'-phosphorylated mutagenic and nonphosphorylated auxiliary oligonucleotides and a specific combination of enzymatic procedures including 'nick-translation' as a key step. The method efficiency was demonstrated by constructing the deletion-insertion mutation which creates the consensus Pribnow box in a promoter-testing plasmid. The yield of the target mutation was up to 85-95%.
Drutsa
V L
VL
A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, USSR.
Kaberdin
V R
VR
D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004270:DNA, Circular / Q000235:genetics* / Q000302:isolation & purification; D005090:Exodeoxyribonucleases; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D015183:Restriction Mapping
D004270:DNA, Circular; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes