Deletion of cagA gene of Helicobacter pylori by PCR products

Figure 1 Strategy for constructing fragments with chloramphenicol resistance gene and gene allelic replacement by PCR. Primers P1-P2, P3-P4 and C1-C2 were amplified and each PCR products were purified. Each three PCR products were mixed and amplified by primer P1 and P4. Then, the PCR product P1-4 was acquired containing three fragments.

Figure 2 PCR amplification for constructing fragments with chloramphenicol resistance gene and gene allelic replacements. The length of cagA/P1-4, a 1.7-kb fragment, is equal to the sum of the length of cagA/P1-2, cagA/P3-4, and cam.

Figure 3 A: Result of PCR amplified for diagnosing homologous recombination strains of cagA gene. Lanes 1-12 are 12 clones selected from chloramphenicol-resistant plates. Control is 26695 wild type. Twelve clones were all negative for diagnostic primers B1-2 while control is positive, suggesting that the flanking region of cagA gene of all the selected clones was deleted; B: Result of PCR amplified for positive control of homologous recombination strains of cagE gene. Lanes 1-12 are 12 clones selected from chloramphenicol-resistant plates. Control is 26695 wild type. Twelve clones were all positive of cagE gene; C: Result of Southern blot for diagnosing homologous recombination strains of cagA gene. Lanes 1-12 are 12 clones selected from chloramphenicol-resistant plates. Control is 26695 wild type. All the 12 clones except 3, 5, and 9 were positive hybridization with probes of chloramphenicol-resistant gene while control was negative.

Figure 4 Mechanism of allelic fragments recombination used by suicide plasmid. The recombination consists of two steps. In the first step, suicide plasmid with PCR products to be exchanged recombined with one allelic fragment and formed a loop. Then, the suicide plasmid recombined with another allelic fragment.