PRGR: A POSITIVE SELECTION VECTOR SYSTEM FOR DIRECT CLONING OF PCR AMPLIFIED DNA FRAGMENTS

摘要

The present invention describes a positive selection vector based on insertional reconstruction of a reporter gene or of a regulatory gene controlling the expression of a reporter gene. The cloning vector carries a reporter gene or a regulatory gene with a dominant mutation at its 3' or 5' end thus rendering the reporter or the regulatory gene protein functionally inactive. A primer carrying a nucleic acid sequence that corrects the mutation is used during PCR amplification of a targeted nucleic acid sequence, and the amplified DNA fragment is then ligated to the cloning vector thus reconstructing the wild-type reporter or regulatory gene. Two positive selection vectors pRGR1Ap and pRGR2Ap have been constructed for use in a positive selection cloning method. Use of pRGR1Ap and pRGR2Ap greatly reduce exonuclease-induced false positive clones.