Rapid Molecular Technique for Clonig of 5' Flanking Region of Potato Gene

Abstract

By conventional way, the promoter region of any gene can be obtained by screening of a huge number of overlapping recombinant clones of a genomic library with the help of a suitable nucleic acid probe. Usually, bacteriophage-based vectors are used for construction of eukaryotic genomic library. But this process of screening is laborious, time-consuming and cost-intensive. On the other hand, a number of PCR-based molecular techniques are also used to amplify DNA outside a region of known sequence. The present thesis work mainly focused on a rapid PCR-based molecular technique namely Single Specific Primer PCR (SSP-PCR) for cloning of 5’ flanking region of any gene of interest. It is one of the faster molecular techniques that can be employed for cloning of promoter region without resorting to conventional cloning procedures. As a case study we followed a cDNA sequence encoding soluble acid invertase from potato (Solanum tuberosum L.) as available in the database. For SSP-PCR, two oligonucleotide primers were designed based on this cDNA sequence where one served as single specific primer. Similarly pUC19 vector-based forward and reverse primers were also designed. Two primer pairs were employed keeping the single specific primer as common along with one vector-specific primer. Partially digested (using EcoRI enzyme) overlapping potato DNA fragments ligated into pUC19 vector were used as template. The size of the amplicon was approx. 1.0 kb. This amplicon was further processed & cloned into the SmaI site of pUC19 vector. A few recombinant plasmids were obtained which were characterized further through restriction analysis as well as PCR technique using internal gene-specific primer. The genomic inserts correspond to the 5’ flanking region of the soluble acid invertase gene as supported by the above results that can be confirmed further by sequencing only. Similarly, Sau3AI digested potato DNA fragments were used in the SSP-PCR as mentioned above. Few clones were also obtained that are yet to be characterized. The molecular technique SSP-PCR appears to be quite promising as experienced from the present study.