Cloning and sequencing of the junction fragment of dystrophin gene with exons 3 to 5 deletion / 南方医科大学学报
Journal of Southern Medical University
;
(12): 757-759, 2006.
Article
in Chinese
| WPRIM
| ID: wpr-282924
ABSTRACT
<p><b>OBJECTIVE</b>To study the mechanisms of dystrophin gene deletion by cloning and sequencing the junction fragment of dystrophin gene with exons 3 to 5 deletion.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>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.</p><p><b>CONCLUSION</b>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.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pathology
/
Molecular Sequence Data
/
Base Sequence
/
Polymerase Chain Reaction
/
Exons
/
Dystrophin
/
Cloning, Molecular
/
Sequence Analysis, DNA
/
Gene Deletion
/
Chromosome Breakage
Limits:
Adult
/
Humans
/
Male
Language:
Chinese
Journal:
Journal of Southern Medical University
Year:
2006
Type:
Article
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