Objective: To clone and analyze Bacillus thuringiensis gene fragments isolated by restriction digest PCR (RD-PCR).
Method: Specific primers were designed to amplify the genes of Bacillus thuringiensis israelensis (Bti), and the PCR products were classified and re-amplified by RD-PCR to obtain the fragments for subsequent purification and cloning into the pMD18-T vectors, followed by rapid identification. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced.
Results: Sequence analysis showed that all the fragments amplified were Bti genes.
Conclusion: RD-PCR is reliable in breaking down large gene fragments into confined and shorter gene fragments for preparing microarray probes.