Development of a versatile cassette for directional genome walking using cassette ligation-mediated PCR and its application in the cloning of complete lipolytic genes from Bacillus species

Since the invention of the PCR technology, adaptation techniques to clone DNA fragments flanking the known sequence continue to be developed. We describe a perfectly annealed cassette available in almost unlimited quantities with variable sticky-and blunt-end restriction enzyme recognition sites for efficient restriction and ligation with the restricted target genomic DNA. The cassette provides a 200-bp sequence, which is used to design a variety of cassette-specific primers. The dephosphorylation prevents cassette self-ligation and creates a nick at the cassette: target genome DNA ligation site suppressing unspecific PCR amplifications. We introduce the single-strand amplification PCR (SSA-PCR) technique where a lone known locus-specific primer is firstly used to enrich the targeted template DNA strand resulting in significant PCR product specificity during the second round conventional nested PCR. The distance between the known locus-specific primer and the nearest location of the restriction enzyme used determined the length of the obtained PCR product. We used this technique to walk downstream into the isochorismatase and upstream into the hypothetical conserved genes flanking the mature extracellular lipase gene from Bacillus licheniformis. We further demonstrated the potential of the technique as a cost-effective method during PCR-based prospecting for novel genes by designing “universal” degenerate primers that detected homologues of Family VII bacterial lipolytic genes in Bacillus species. The cassette ligation-mediated PCR was used to clone complete nucleotide sequences encoding functional lipolytic genes from B. licheniformis and Bacillus pumilus.