[Cloning and sequencing of the junction fragment of dystrophin gene with exons 3 to 5 deletion].
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.


CONCLUSIONS
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.
Zhong
Min
M
Department of Neurology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. pomelomin@hotmail.com
Pan
Su-yue
SY
Lu
Bing-xun
BX
Jiang
Li
L
Li
Wei
W
D000328:Adult; D001483:Base Sequence; D019457:Chromosome Breakage; D003001:Cloning, Molecular; D016189:Dystrophin / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D017353:Gene Deletion; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009136:Muscular Dystrophies / Q000235:genetics* / Q000473:pathology; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
D016189:Dystrophin