Positive selection vectors for high-fidelity PCR cloning

The power of PCR cloning of a target DNA fragment is limited by polymerase-induced mutations. While high-fidelity PCR products can be achieved by reducing the number of PCR cycles, the cloning of the very small amount of DNA thus amplified should give only a few recombinant clones (carrying an insert), which would be very difficult to screen from thousands of background false-positive clones generated by all the currently available vectors, including the positive selection vectors. False-positive clones are mostly generated by the recircularization of linearized vectors that have lost some bases at their ends due to digestion with contaminating exonuclease activities present in restriction enzymes, ligases, polymerases, and other reagents. To overcome this problem, two positive selection vectors, pRGR1Ap and pREM5Tc, have been developed, based on the principles of reporter gene reconstruction and regulatory element modulation, respectively. A PCR primer carrying a vector-specific sequence at its 5' end is used in PCR. When the resultant PCR products are ligated to the specific vector, an antibiotic resistance gene is expressed, thus donating positive selection capability to the harboring cells in a specific selection medium. These vectors cloned PCR fragments generated from less than a femtomole quantity of Escherichia coli genomic DNA after only three cycles of PCR amplification, thus greatly reducing the number of recombinant clones containing polymerase-induced mutations.