Figure 1

Principle of REPLACR-mutagenesis and primer design strategy for sequence substitution, addition or deletion.

(a) Primers containing the desired mutation are designed to target a specific region in the original vector. A high-fidelity polymerase is used to generate a linear PCR product such that both the ends contain overlapping sequences for recombination. Bacteria expressing the recombineering proteins (Redγ, β, α and RecA) are transformed with the PCR product. Recombination takes places inside the bacteria thereby yielding a circular plasmid containing the desired mutation. Bacterial colonies are then screened for the correct clone by PCR and sequencing. (b) Forward and reverse primers contain the desired addition/substitution as a part of homology regions needed for recombination, besides containing a 3′ extension for effective template binding. The homology region (17 bp or more) for substitutions also contains the desired nucleotide change. (c) For generating deletion mutants, forward primer contains the sequence adjoining the sequence to be deleted. The reverse primer however contains a sequence homologous to the forward primer and the adjoining sequence in the vector.