We actually had tried out this method bout 20 years ago, when the H. influenzae genome sequence first became available, but after some experiments we decided that cloning was usually more reliable. But now I'm in a situation where cloning is being very unreliable indeed, enough so that I can't bring myself to try again. So instead I'm going to try the direct-transformation method.
- In two independent PCR reactions, amplify the left and right genome segments flanking the gene to be deleted (the antitoxin gene). These segments need to be long enough to later allow efficient homologous recombination of the final construct into the chromosome.
- Separately amplify a Spectinomycin-resistance cassette (SpcR), using primers designed with tails complementary to the inner ends of the two genome fragments.
- Remove all primers from these PCR products and mix them together in a PCR reaction mix with no added primers.
- Do one cycle of strand melting, strand annealing (of the primer tails), and strand extension by Taq. This produces two mid-length fragments that both contain the SpcR segment.
- Do another cycle of strand melting, strand annealing (this time of the full SpcR segment), and strand extension by Taq. This produces one full-length fragment containing both genome segments and the SpcR segment.
- Add the outermost genome primers (left primer of the left segment and right primer of the right segment) and carry out a normal PCR amplification.
- Transform the resulting fragment into competent A. pleuropneumoniae cells.
- Select for SpcR and confirm the new genotype by PCR.
Now I need to dig into the sequence file and primer files to figure out whether I can reuse primers we already have, and to design the new SF and SR primers with the appropriate tails.