Transitory selection markers facilitate DNA mutagenesis with recombineering

Abstract

Site-directed mutagenesis with DNA oligos is a powerful tool to generate subtle alterations in DNA. Taking advantage of strand replacement during lambda-red recombineering, mutations on DNA (donor) can be introduced into another DNA construct (acceptor) via targeting.  We describe a novel approach that takes advantage of the specificity of homologous recombination and the convenience of positive selection to insert mutations into existing DNA constructs.  With simple procedures, we generated a “transitory” selection marker that is used to carry modifications into acceptor DNA and then removed by enzyme manipulation, leaving only the desired mutation without any scar.  Overall, this method of targeting mutagenesis via recombineering provides a precision manipulation of DNA and avoids the extensive PCR reaction.  Not only can this method be used to make point mutations, truncations or Epitope-tags, but it can also be used to import existing mutations, generate in-frame fusion, or repair missing components.