A protocol for detection of mitochondrial DNA deletions: characterization of a novel deletion.
OBJECTIVE
To develop a protocol capable of identifying deletions in mitochondrial DNA and use it to identify the breakpoints of a mtDNA deletion in a patient with chronic progressive external ophthalmoplegia (CPEO).


METHODS
Deletions in mtDNA were identified by a combination of long range PCR and Southern blotting. The precise breakpoints were determined by automated DNA sequencing.


RESULTS
A series of DNA samples from patients with suspected mitochondrial disease was subjected to a protocol, which combines long range PCR and Southern blotting. We found a unique deletion in a patient with CPEO and we identified the precise location of this deletion through DNA sequencing.


CONCLUSIONS
Long range PCR has the advantages of speed, minimal samples requirements, and sensitivity. Southern blotting is better able to evaluate heteroplasmy and detect duplications. We suggest a protocol that enables us to identify precisely the breakpoints in a unique mutation of mtDNA in a patient with CPEO.
Coulter-Mackie
M B
MB
Department of Pediatrics, University of British Columbia, Vancouver, Canada. marioncm@interchange.ubc.ca
Applegarth
D A
DA
Toone
J R
JR
Gagnier
L
L
D000293:Adolescent; D001763:Blepharoptosis / Q000235:genetics; D015139:Blotting, Southern / Q000379:methods*; D004272:DNA, Mitochondrial / Q000032:analysis*; D006801:Humans; D007625:Kearns-Sayre Syndrome / Q000235:genetics; D008297:Male; D017237:Mitochondrial Encephalomyopathies / Q000235:genetics; D009886:Ophthalmoplegia / Q000235:genetics*; D017246:Ophthalmoplegia, Chronic Progressive External / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017384:Sequence Deletion
D004272:DNA, Mitochondrial