Abstract: The present invention relates to the development of 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 mutation 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 said vector thus reconstructing the wild-type reporter or regulatory gene.

Abstract: The present invention describes a methodology for generating high fidelity PCR products, and also cloning of such high fidelity PCR products in a suitable vector. Generation of polymerase-induced mutant fraction of target sequences during PCR amplification is linearly proportional to the number of doublings of the target sequences. Thus the high fidelity PCR products are generated by minimizing the number of doublings of the target nucleic acid sequences during PCR amplification. Minimization of number of doublings of the target sequences is achieved by reducing the number of cycles of PCR amplification of the target sequences. The high fidelity PCR products thus obtained are then cloned into a suitable vector. As an example, a 960 bp target sequence from E. coli DNA was PCR-amplified only for 3 cycles, and it was then directly cloned into a positive selection cloning vector pRGR2Ap.