سیدجواد حسینی

Identifying unknown sequences adjacent to a known sequence has many applications, such as identifing site of the transgenes, finding the location of the transposons, to discover regulatory factors of the genes or the isolation of the promoter for the creation of specific vectors of the species. There is several methods for identifying these unknown sequences, which are often classified into two categories, cutting-dependent and primer-dependent. One of these methods is provided by Clontech Company in form of the Genome Walker Kit. In this kit, the Blunt end making enzymes have been used. Because the addition of Blunt ends has its own problems, using of enzymes producing the same sticky ends was considered.In this study, an unknown sequence of beta-actin promoter was used to study this optimization. By using the Bgl II enzyme, two pieces of 300bp and 1200bp were obtained, which was with mild smear. The results obtained in this study were not good enough and it seems that the method used has not been able to prevent amplification of non-specific sequences and there were always lots of Fragments in the final stage. It seems that with the change in the Adaptor structure in future research, the process of Isolation could be made more qualitatively.