Bridge-overlap-Extension PCR method for constructing chimeric genes

Abstract

We understand that this bridge-overlap-extension PCR approach can have several applications, such as insertion of epitope tags, nuclear localization signals, secretory or proteolytic signals, insertion of DNA-binding regions or other functional elements into proteins of interest.The sequence element to be inserted can be of small size or long both being within the limits of efficient oligonucleotide synthesis. A distinct feature of this method is that by using overlapping oligonucleotides and bringing about a bridging step, we carry out the synthesis of a longer primer that contains an overlap with the hybrid PCR product, in the absence of any wild-type template. This helps in avoiding the use of template DNA and, therefore, the problem of re-annealing by oligonucleotides to wild-type template during the PCR steps altogether. The success of the technique is indicated by the 100% efficiency of obtaining the correct clones. Moreover, a high level of expression of precursor (fusion of P-factor and SK) and mature SK was observed when the hybrid gene construct, was cloned into an expression vector and transformed in suitable strains of S-pombe- Therefore, this new approach could serve as an alternative method for construction of chimeric genes.