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Overlap-extension PCR is a method for splice of gene segments to produce focused fragments for constructing recombinant plasmid, but its complexity limits its application. To simplify the protocol and to improve the effectiveness, we employed gradient temperatures to replace the single annealing temperature in the thermo-cycling program, and optimize the templates ratio. The concentration of each fragment was adjusted to 10 ng µl . Fragment concentration ratio was the inverse of the fragment size ratio. The products of fused segments were 2000-5000 bp in length using the revised one-step method. This method splices effective two or more fragments to fused gene and produce recombinant plasmid.