Scientists Use Recombineering to Initiate Site-Directed Mutagenesis

Site-directed mutagenesis (SDM) is used to study the resulting translation product for functional characterization. Meanwhile, homologous recombination (HR) is a process where homologous DNA fragments exchanges nucleotides to repair DNA breaks. This mechanism was eventually used to modify plasmids and is now called recombineering.

The research team led by Ashutosh Trehan from the University of Turku in Finland presents a single-step method, called REPLACR-mutagenesis (Recombineering of Ends of linearised PLAsmids after PCR), for generating site-directed modifications in plasmids by in vivo recombineering. REPLACR-mutagenesis only involves inserting PCR products into bacteria expressing recombineering proteins.

In REPLACR mutagenesis, primers with the desired mutation are designed to target a specific region in the original vector. A linear PCR product with both ends containing similar sequences is then generated. Bacteria expressing the recombineering proteins are transformed with the PCR product and recombination takes place inside the bacteria, yielding a circular plasmid containing the desired mutation.

For more on this promising technology, read the full article in Nature.