Using Recombineering to Generate Point Mutations: The Oligonucleotide-Based “Hit and Fix” Method

Ability to manipulate the genome or design genes with desired mutation is critical for functional studies. Recombineering has made genetic manipulation of large genomic fragments very feasible and efficient. In the bacteriophage lambda-based recombineering system, three prophage genes, exo, bet, and gam, under the control of a temperature-sensitive lambda cI-repressor, provide the recombination function. The high efficiency of recombineering by oligonucleotides allows generation of subtle alterations in the bacterial chromosomal DNA as well as episomal DNA. We describe here a two-step "Hit and Fix" method, in which a short heterologous sequence is inserted to the target site first (Hit) and this sequence is replaced with the desired mutation in the second step (Fix). Insertion and replacement of the heterologous sequence allows screening of the recombinant clones by PCR or colony hybridization.