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Updated: Aug 3, 2026
07:11
Homemade Site Directed Mutagenesis of Whole Plasmids
Published on: May 11, 2009
A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and
M Sugimoto1, N Esaki, H Tanaka
1Institute for Chemical Research, Kyoto University, Japan.
Analytical Biochemistry
|June 1, 1989
Summary
This study introduces a straightforward method for oligonucleotide-directed mutagenesis using double-stranded DNA. The technique rapidly prepares templates and achieves high-frequency gene mutation, simplifying molecular biology research.
Area of Science:
- Molecular Biology
- Genetic Engineering
Background:
- Oligonucleotide-directed mutagenesis is crucial for genetic studies.
- Efficient template preparation and high mutation yield are desired.
Purpose of the Study:
- To develop a simple and efficient method for oligonucleotide-directed mutagenesis.
- To enable rapid template preparation and high-frequency mutation of double-stranded DNA.
Main Methods:
- Utilized double-stranded plasmid DNA as a template.
- Prepared template DNA via cell lysis and alkali denaturation.
- Employed phosphorothioate-modified nucleotides for chain elongation at 37°C.
- Applied selective digestion using NciI and exonuclease III.
Main Results:
- Achieved a high frequency of the desired mutated gene (approximately 70%).
- Demonstrated a simple and rapid template preparation process.
Conclusions:
- The developed method is simple, efficient, and rapid for oligonucleotide-directed mutagenesis.
- This technique facilitates high-yield generation of mutated genes from double-stranded DNA templates.
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