P3 site-directed mutagenesis: An efficient method based on primer pairs with 3'-overhangs

Area of Science:

• Molecular Biology
• Biotechnology
• Genetic Engineering

Background:

• Site-directed mutagenesis is crucial for protein and plasmid engineering.
• Existing methods like QuickChange have limitations in efficiency and accuracy.
• Achieving 100% mutagenesis efficiency remains a technological challenge.

Purpose of the Study:

• To optimize a novel site-directed mutagenesis strategy using 3 -overhang primers.
• To develop a more efficient and economical method for generating various mutations.
• To evaluate the performance of the new method across diverse expression vectors.

Main Methods:

• Systematic optimization of primer design, DNA polymerases, and PCR parameters.
• Extensive testing on 12 mammalian expression vectors (7.0–13.4 kb) and two SARS-CoV-2 spike protein vectors.
• Comparison of the new method against the QuickChange method for efficiency and mutation generation.

Main Results:

• The optimized method achieved an average efficiency of approximately 50%, with some instances reaching or nearing 100%.
• Substantially increased success rate and reduced engineering time compared to the QuickChange method.
• Successful mutagenesis on large expression vectors encoding epigenetic regulators and viral proteins.

Conclusions:

• A novel, efficient, and economical site-directed mutagenesis method has been developed.
• The method demonstrates significant improvements over existing techniques for plasmid engineering.
• Further refinement is needed to address challenges with extremely GC-rich sequences for universal application.