Generation of a PCR amplicon for insertion/deletion mutagenesis of the pilA gene. In Panel A, the pilA gene from NTHi strain 2019 is shown. Homology arm 1 (H1) contains 50 base pairs of DNA 5' to and including the ATG start codon of pilA. Homology arm 2 (H2) contains the complement of the last 21 base pairs of the pilA gene and 29 base pairs immediately downstream of the pilA gene. In Panel B, the template region of pRSM2832 is shown. Primer H1P1 contains the sequence from H1 and 20 base pairs of DNA homologous to the 5' end of the cassette (P1). Primer H2P2 contains the sequence from H2 and 20 base pairs of DNA homologous to the 3' end of the cassette. PCR amplification of pRSM2832 with primers H1P1 and H2P2 (primers 5 and 6) yielded the product shown in Panel C.