A protocol for detection of mitochondrial DNA deletions: characterization of a novel deletion

• Published in: Clinical biochemistry Vol. 31; no. 8; pp. 627 - 632
• Main Authors: Coulter-Mackie, Marion B, Applegarth, Derek A, Toone, Jennifer R, Gagnier, Liane
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.1998
• Subjects: Adolescent; Blepharoptosis - genetics; Blotting, Southern - methods; DNA, Mitochondrial - analysis; Humans; Kearns-Sayre Syndrome - genetics; long range PCR; Male; mitochondrial DNA deletions; Mitochondrial Encephalomyopathies - genetics; Ophthalmoplegia - genetics; Ophthalmoplegia, Chronic Progressive External - genetics; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Sequence Deletion; Southern blotting; mitochondrial DNA deletions; Southern blotting; long range PCR
• ISSN: 0009-9120; 1873-2933
• DOI: 10.1016/S0009-9120(98)00074-5

Objectives: 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). Design and 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 sample 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.