Abstract: In order to assemble large fragments of DNA sequence efficiently, the study modified a advanced method, successive anneal overlap extension PCR, base on the traditional overlap extension PCR. The method of successive anneal overlap extension PCR lengthens the primer sequences corresponding to overlapping region between two fragments and uses successive annealing ranging from 3 to 6℃ in each single PCR cycles of the second PCR process. In this study, we assembled two large DNA fragments, ω3（2）and HCT, using this advanced method. A single band was obtained with the PCR product and the sequence was proved to be right after sequencing repectively, which would contribute to next experiments. This modified method has higher specificity and sensitivity, and is called successive anneal overlap extension PCR.