Adeno-associated virus type 5 (AAV5), which is distinct from the other serotypes of AAV, has attracted considerable interest as a premier gene delivery vector. As do the other serotypes, AAV5 contains its 4.7 kb-sized, single-stranded genome flanked with inverted terminal repeats (ITRs) in a hairpin conformation, which serves frequently as pause and arrest sites for DNA polymerases during PCR. To amplify the full-length of the AAV5 genome in single step, we established a shuttled, long and accurate PCR (LA-PCR) procedure in the present study. Furthermore, helper oligonucleotides, which hybridize with the palindromic sequence elements in ITR, were designed and employed in PCR to prevent the formation of hairpin structures by highly GC-rich ITRs. Consequently, a 4.7 kb-sized PCR product was amplified successfully, and cloned into a pBluescript II KS(+) plasmid. Six plasmids, harboring the full-length AAV5 genome, rescued wild type AAV5 viruses on transfection to HeLa and HEK 293 cells, which were co-infected with helper adenoviruses. Western and Southern blot analyses supported further the fact that the pAAV5 plasmids harbored the full-length AAV5 genome. The PCR method described in this study is applicable for the cloning of genomes containing variable palindromic structures, in addition to AAV genomes of other serotypes.