A novel polymerase chain reaction (PCR) for rapid isolation of a new rbcS gene from Lemna minor

Abstract

This study developed a novel polymerase chain reaction (PCR) method, ligation-mediated self-formed panhandle PCR, for gene or chromosome walking. It combined the advantages of ligation-mediated PCR in its specificity and of panhandle PCR in its efficiency. Self-formed panhandle PCR was used for a new rbcS gene walking to isolate 3’ downstream and 5’ upstream sequence; 1292 bp DNA rbcS gene was obtained via 3’ walking of Lemna minor gemonic DNA and 5’ upstream sequence of the new rbcS gene with a length of 1543 bp was isolated from L. minor via self-formed panhandle PCR. A novel rbcS gene with the size of 2835 bp, which was confirmed by nested-PCR, was obtained by ligation-mediated self-formed panhandle PCR. Ligation-mediated self-formed panhandle PCR was simple and efficient and should have broad applications in the isolation of unknown sequences in genomes.

Keywords: Chromosome walking, Lemna minor, polymerase chain reaction (PCR), rbcS gene, self-formed panhandle.

African Journal of Biotechnology, Vol 13(45) 4282-4288