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).
Keywords: Haemophilus influenzae, Gene deletion, Mutants, Mutagenesis, Molecular microbiology
Figure 1. Schematic representation of the Megaprimer PCR method. Step A1. The flanking regions (~1,000 bp) of the target gene are amplified by PCR (Primer pair L1 + L2 and R1 + R2). The L2 and R2 primers have extensions with homology to the antibiotic resistance cassette. Step A2. The antibiotic resistance cassette is amplified by PCR (primer pair L + R). Step B. The three PCR products of the first PCR reactions are combined. The flanking regions anneal to the antibiotic resistance cassette and one large PCR product is formed.
Materials and Reagents
Equipment
Procedure
Notes
Acknowledgments
This protocol is adapted from a previously published paper: Langereis et al. (2013).
References
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