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Related Experiment Videos

Improved oligonucleotide site-directed mutagenesis using M13 vectors.

P Carter, H Bedouelle, G Winter

    Nucleic Acids Research
    |June 25, 1985
    PubMed
    Summary
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    This study introduces a coupled priming technique for efficiently constructing mutations in M13 vectors. The method uses selectable markers to improve mutant yields, achieving up to 70% success in genetic engineering.

    Area of Science:

    • Molecular Biology
    • Genetic Engineering
    • Microbiology

    Background:

    • Site-directed mutagenesis is crucial for genetic research.
    • Existing methods for M13 vector mutagenesis can be inefficient.
    • Novel strategies are needed to improve the construction of specific mutations.

    Purpose of the Study:

    • To develop an improved method for constructing mutations in M13 vectors.
    • To enhance the efficiency and yield of mutagenesis using synthetic oligonucleotides.
    • To introduce a selectable marker system for efficient mutant selection.

    Main Methods:

    • Cloning DNA into a novel M13 vector with a selectable marker (EcoK, EcoB, or amber mutation).
    • Employing a "coupled priming" technique with two primers to introduce mutations and eliminate the marker.

    Related Experiment Videos

  • Transfecting heteroduplex DNA into repair-deficient E. coli strains for selective marker removal.

    • Main Results:

    • Successfully constructed over 50 mutants using the described method.
    • Achieved excellent yields of desired mutants, up to 70%.
    • Demonstrated the advantage of EcoK/EcoB markers for stepwise mutagenesis and cycling selection.

    Conclusions:

    • The coupled priming technique offers an efficient approach for M13 mutagenesis.
    • Selectable markers significantly improve mutant yields and streamline the selection process.
    • Alternative strategies using long oligonucleotides are viable for constructing multiple mutations in close proximity.