Generation of Non-typeable Haemophilus influenzae Directed Gene Deletion Mutants

Abstract

Directed deletion mutants in non-typeable Haemophilus influenzae can be made by allelic exchange of the target gene with an artificial DNA construct in which an antibiotic resistance cassette is placed between two ~1,000 bp DNA sequences that are identical to the 5' and 3' flanking regions of the target gene. The artificial DNA construct that is required for this mutagenesis is synthesized by the so-called Megaprimer PCR method (Figure 1).