Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific Surveyor Nuclease

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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.

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Figure 1
Figure 2: Human mtDNA map showing location of the 17 amplicons, labeled A–P.
Figure 3: Agarose gel showing the PCR amplification of 4 out of 17 mtDNA amplicons and “Surveyor control”.
Figure 4: Identification of mtDNA mismatches following Surveyor Nuclease digestion.
Figure 5: Example of result that requires the confirmation of the mutation by RFLP analysis.
Figure 6: Schematic representation of RFLP principle (see Box 1).

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Acknowledgements

We thank B Chafino, A Blombou, S Charabot and A Figueroa for technical help. We also thank F Stefani and N Neckelmann from Transgenomic for helpful discussions. This work was made possible by grants from CHU Nice (CPR UF 699), France.

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Correspondence to Veronique Paquis-Flucklinger.

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The authors declare no competing financial interests.

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Bannwarth, S., Procaccio, V. & Paquis-Flucklinger, V. Rapid identification of unknown heteroplasmic mutations across the entire human mitochondrial genome with mismatch-specific Surveyor Nuclease. Nat Protoc 1, 2037–2047 (2006) doi:10.1038/nprot.2006.318

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