Kunkel Mutagenesis
BioInnovatise Cloning Team
Updated May 29, 2025
Molecular Cloning & Mutagenesis Projects Completed
Viral Vector Packaging Solutions Delivered
Plasmid DNA Preparations Successfully Produced
Kunkel mutagenesis is a type of site directed mutagenesis for precision plasmid DNA modifications. It was originally developed for single-stranded phage DNA, modern adaptations make it highly effective for plasmid mutagenesis. Our plasmid DNA team performs Kunkel mutagenesis for researchers who are typically working on larger plasmid DNA constructs (>10 KB) and want higher mutation efficiency.
At BioInnovatise, we preform all mutations using the specific technique requested by the researcher in order to achieve the mutation they are looking for on a plasmid DNA construct.
Our plasmid DNA team explains a little bit about Kunkel mutagenesis, why researchers may prefer it to PCR / transposon mutagenesis, and what you need to know before starting a production.
Site Specific Kunkel Mutagenesis And Its Advantages
During site specific mutation productions, the Kunkel mutagenesis technique takes place through selective strand degradation starting with plasmid template preparation:
- Plasmid Template Preparation: The plasmid is transformed into a dut⁻ ung⁻ E. coli strain that incorporates uracil instead of thymine and lacks uracil repair mechanisms.
- Uracil-Containing Template: After growth, the plasmid DNA contains uracil residues throughout one strand.
- Primer Annealing: A synthetic oligonucleotide containing the desired mutation is annealed to the uracil-containing plasmid template.
- In Vitro Synthesis: DNA polymerase extends the primer around the entire plasmid, creating a heteroduplex with one uracil-containing strand and one normal strand.
- Transformation And Repair: The heteroduplex plasmid is transformed into repair-proficient E. coli, which selectively degrades the uracil-containing strand and repairs using the mutant strand as template
- (Optional) Plasmid Preparation and Purification: After the mutation has been verified via sanger sequencing, our plasmid DNA team can produce larger volumes of the plasmid DNA for downstream applications.
The advantages of kunkel mutagenesis center on its ultra specific site-specific targeting for mutations. The exact position and type of mutation is controlled by primer design, allowing researchers to change specific codons in cloned genes, modify regulatory sequences, create or destroy restriction sites, and insert or delete small sequences.
Kunkel Mutagenesis vs Transposon and PCR Mutagenesis
Kunkel mutagenesis does not rely on PCR or transposons to initiate sequence mutations. Kunkel uses primer extension on prepared templates rather than thermal cycling as PCR mutagenesis does. Transposon mutations create random insertions in plasmids via transposase proteins while Kunkel mutations are precise and deliberative.
Not sure which mutation technique is right for your research? Don’t worry, reach out to our plasmid DNA team and we will determine which mutagenesis technique is right for your plasmid DNA requirements.
Plasmid DNA Library Construction Using Kunkel Mutagenesis
Let’s get started! To begin your Kunkel mutagenesis production we require the following information when submitting your project details.
- The complete sequence of the template (the target) and indicated sites for mutation.
- 3 µg of wildtype plasmid DNA.
- Your selection of selection markers, promoters, and other regulatory elements.
Precision medicine research and development progresses everyday, and with it, the need for high-integrity mutant plasmid DNA.
Want to learn more about the latest in mutagenesis? Our colleagues at ScienceDirect, the American Society for Biochemistry and Molecular Biology, and Genetic Engineering and Biotechnology News continuously collect and publish the latest information on genetic mutation research.
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