A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide.

M Sugimoto; N Esaki; H Tanaka; K Soda

doi:10.1016/0003-2697(89)90134-6

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A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide.

M Sugimoto·N Esaki·H Tanaka·K Soda

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Abstract

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%).

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Amino Acid Sequence Base Sequence DNA DNA Restriction Enzymes Deoxycytosine Nucleotides Deoxyribonucleases, Type II Site-Specific Exodeoxyribonucleases Molecular Sequence Data Mutation Nucleic Acid Hybridization Oligonucleotides Plasmids Thionucleotides Journal Article

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M Sugimoto, N Esaki, H Tanaka, K Soda. 1989. A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide.. https://doi.org/10.1016/0003-2697(89)90134-6

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