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

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 PGR 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 PGR 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. (C) 1998 The Canadian Society of Clinical Chemists.