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Updated: Jun 20, 2025
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Homemade Site Directed Mutagenesis of Whole Plasmids
Published on: May 11, 2009
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Single-round QuikChange PCR for engineering multiple site-directed mutations in plasmid DNA.
Yunxiang Li1, Mileina Pinones1, Alexis Breeland2
1Division of Chemistry and Biochemistry, Texas Woman's University, Denton, TX, 76204, USA.
Analytical Biochemistry
|July 17, 2024
Summary
This study introduces a rapid method for simultaneous gene editing at multiple sites using polymerase chain reaction (PCR). The technique efficiently creates diverse mutations, accelerating genetic engineering research.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- Mutational studies are essential for gene engineering.
- Existing mutagenesis strategies aim to improve efficiency.
- Need for faster, multi-site mutation introduction methods.
Purpose of the Study:
- To develop a simple, time-saving method for simultaneous mutagenesis at discrete sites.
- To enable multiple nucleotide insertions, deletions, and replacements in a single polymerase chain reaction (PCR) round.
- To facilitate the creation of mutant libraries for various research applications.
Main Methods:
- Utilized plasmid as a template.
- Employed compatible oligonucleotide primers based on the QuikChange strategy.
- Performed simultaneous mutagenesis in one round of PCR.
Main Results:
- Successfully introduced multiple nucleotide insertions, deletions, and replacements.
- Achieved longest insertion of 28 bp and deletion of 16 bp.
- Enabled up to 4 discrete nucleotide replacements (≤4 bp mismatch) simultaneously.
- Identified clones with all desired mutations typically by screening 5 colonies.
Conclusions:
- The developed method is simple, fast, and efficient for simultaneous multi-site mutagenesis.
- It allows for the creation of combinatory mutant libraries via saturation mutagenesis.
- This approach is promising for advancing research in protein biochemistry, forward genetics, and synthetic biology.
