[Cloning and sequencing of junction fragment with exon 51 deletion of Dystrophin gene].

ABSTRACT

To study the mechanism of Dystrophin gene deletion, we obtained the deletion junction fragment of exon 51 by inverse PCR and analyzed the sequence characteristic of breakpoints and deletion junction fragment. After the full sequence of intron 51 was finished, the rough site of breakpoint in intron 51 of a DMD patient with exon 51 deletion was detected by PCR with 9 pairs of primers spaced every 3-5 kb in intron 51. Then the junction fragment was amplified by nested inverse PCR. After sequencing the junction fragment, the 3' breakpoint and partial sequences of intron 50 were determined by comparing with the normal sequences in intron 51. The primer was designated to sequence intron 50 according to the sequence of junction fragment, and then the 5' breakpoint was determined. A total of 1,614 bp in intron 50 was sequenced. The 5' and 3' breakpoints were located in the THE-1 internal sequence (Transposon-like Human Element, THE) and L2 sequence respectively. There are 3 bp junctional homology and no errors near the junction point. This is the second report that the deletion breakpoint located directly in THE-1 sequence studied at the DNA level. We here firstly reported that there is a THE-1 sequence in intron 50. THE-1 and non-homologue end joining repair mechanism may be associated with the Dystrophin gene deletion.