Abstract: Objective To study the mechanisms of dystrophin gene deletion by cloning and sequencing the junction fragment of dystrophin gene with exons 3 to 5 deletion. Methods PCR was performed to verify dystrophin gene exons 3 to 5 deletion in a patient with Duchenne muscular dystrophy. A PCR-based genome-walking method was used to localize the breakpoint in introns 2 and 5, and the deletion-junction fragment was directly amplified by PCR approach with forward and reverse primers annealing to a DNA sequence as close as possible to the breakpoint in the introns 2 and 5. The sequencing result of the deletion-junction fragment was compared with the normal intron sequences. Results A sequence of 2113 bp containing the junction fragment was obtained. The 5’ breakpoint was located in SINE/Alu element of intron 2, and the 3’ breakpoint was located in the unique sequence near the sequence TTTAAA. The breakpoints were associated with a strong topoisomerase Ⅱ cleavage site. A 26-bp fragment was inserted into the breakpoint and formed 3 duplications (GGCTTATATTTAA) of 13 bp around the deletion-junction fragment. Conclusion Repeat sequence and strong topoisomerase Ⅱ cleavage site around the breakpoint may predispose double-strand DNA breaks and recombination, which, in addition to the nonhomologous end-joining mechanism, may contribute as important factors to the gene deletion.