Seyed Javad Hosseini

Identification of unknown sequences adjacent to a known sequence has various applications, including: Identifying the insertion site of bacteriophages, the place of pairing of transposons, recognition of gene regulatory factors and also the direction of making specific vectors. There are variety of methods to identify unknown sequences, which are often divided into three general categories, depending on the cutting site, depending on the primer, and depending on the extension of the sequence. One of these methods is based on the Genome Walker kit made by Clon-tech. In this kit, enzymes with cutting sites producing smooth ends are used, which have many problems for joining. For this reason, the use of universal degenerated primers was the focus of this research. To evaluate this method, we used gene-specific primers, degenerated primers and human LDL receptor gene as template. According to the results obtained after moving the products on the agarose gel and observing the bands of about 1100 bp, it seems that the method mentioned in this research was able to prevent the proliferation of non-specific fragments and lead us to the target sequence.