Protocol abstract
Nature Protocols 1, - 2037 - 2047 (2006)
Published online: 30 November 2006 | doi:10.1038/nprot.2006.318
Subject Categories: Genomics and proteomics | Nucleic acid based molecular biology
Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific Surveyor Nuclease
Sylvie Bannwarth1, Vincent Procaccio2 & Veronique Paquis-Flucklinger1,3
Abstract
Mitochondrial DNA (mtDNA) mutations are responsible for mitochondrial diseases in numerous patients. But, until now, no rapid method was available for the identification of unknown deleterious point mutations. Here, we describe a new strategy for the rapid identification of heteroplasmic mtDNA mutations using mismatch-specific Surveyor Nuclease. This protocol involves the following three steps: (i) PCR amplification of the entire human mitochondrial genome in 17 overlapping fragments; (ii) localization of mtDNA mismatch(es) after digestion of the 17 amplicons by Surveyor Nuclease; and (iii) identification of the mutation by sequencing the region containing the mismatch. This Surveyor Nuclease-based strategy allows a systematic screening of the entire mtDNA to identify a mutation within 2 days. It represents an important diagnostic approach for mitochondrial diseases that can be routinely used in molecular diagnostic laboratories.
- Department of Medical Genetics, Archet 2 Hospital, CHU Nice, France.
- Center for Molecular and Mitochondrial Medicine and Genetics, University of California, Irvine, California, USA.
- UMR CNRS/UNSA 6543, Medicine School, University of Nice-Sophia Antipolis, Nice, France 0602.
Correspondence to: Veronique Paquis-Flucklinger1,3 e-mail: paquis@unice.fr
