Long Distance-Inverse PCR Technique for Efficient Cloning of Flanking Sequences Adjacent to Known DNA Fragments

Abstract: To avoid the disadvantages of conventional inverse PCR such as short amplification fragments and pseudo-positive products, the long distance-inverse PCR (LD-IPCR) technique was set up as follows. In a 1-ml reaction system, 0.5 μg of DNA fragments excised by a single restriction enzyme was adequately self-looped and ligated. Then the reaction products were used as the templates for LD-IPCR amplification using specific inverse primer pairs of 25～30 nucleotides. It was proven that this modified technique could successfully make it more efficient, convenient as well as more specific in cloning the flanking sequences adjacent to DNA fragments obtained. The flanking sequence amplified by LD-IPCR could reach 16 kb in length.