A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide.
We have developed a simple and efficient method for oligonucleotide-directed mutagenesis with double-stranded (plasmid) DNA as a template. The template was simply and rapidly prepared by cell lysis and the following DNA denaturation with alkali. The chain elongation was performed with phosphorothioate-modified nucleotide at 37 degrees C. After the selective digestion of original DNA with NciI and exonuclease III, the desired mutated gene was obtained at a high frequency (about 70%).
Sugimoto
M
M
Institute for Chemical Research, Kyoto University, Japan.
Esaki
N
N
Tanaka
H
H
Soda
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D003845:Deoxycytosine Nucleotides; D015252:Deoxyribonucleases, Type II Site-Specific; D005090:Exodeoxyribonucleases; D008969:Molecular Sequence Data; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009841:Oligonucleotides; D010957:Plasmids; D013873:Thionucleotides
D003845:Deoxycytosine Nucleotides; D009841:Oligonucleotides; D013873:Thionucleotides; C032559:deoxycytidine thiotriphosphate; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes; C035801:CCSGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific