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Design and Use of a Peptide Nucleic Acid for the Detection of the Heteroplasmic Low-Frequency Melas Mutation in Human Mitochondrial DNA



Diane K. Hancock, Frederick P. Schwarz, F H. Song, L J. Wong, Barbara C. Levin


Most pathogenic human mitochondrial DNA (mtDNA) mutations are heteroplasmic (i.e., mutant and wild-type mtDNA coexist in the same individual) and are difficult to detect when their concentration is a small proportion of that of wild-type mtDNA molecules. We describe a simple methodology to detect low proportions of the single base pair heteroplasmic mutation, A3243G that has been associated with the disease mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) in total DNA extracted from blood. Methods: Three peptide nucleic acids (PNAs) were designed to bind to the wild-type mtDNA in the region of nucleotide position 3243, thus blocking PCR amplification of the wild-type mtDNA while permitting the mutant DNA to become the dominant product and readily discernable. DNA was obtained from both apparently healthy and MELAS individuals. Optimum PCR temperatures were based on the measured ultraviolet thermal stability of the DNA/PNA duplexes. The presence or absence of the mutation was determined by sequencing. Results: In the absence of PNAs, the heteroplasmic mutation was either difficult to detect or undetectable by PCR and sequencing. Only PNA 3 successfully inhibited amplification of the wild-type mtDNA while allowing the mutant mtDNA to amplify. In the presence of PNA 3, we were able to detect the heteroplasmic mutation when its concentration was as low as 0.1% of the concentration of the concentration of the wild-type sequence. Conclusion: This methodology permits easy detection of low concentrations of the MELAS A 3243G mutation in blood by standard PCR and sequencing methods.
Environmental and Molecular Mutagenesis
No. 12


heteroplasmy, human, MELAS, mitchondrial DNA (mtDNA), peptide Nucleic acid (PNA)


Hancock, D. , Schwarz, F. , Song, F. , Wong, L. and Levin, B. (2002), Design and Use of a Peptide Nucleic Acid for the Detection of the Heteroplasmic Low-Frequency Melas Mutation in Human Mitochondrial DNA, Environmental and Molecular Mutagenesis (Accessed July 19, 2024)


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Created August 1, 2002, Updated February 17, 2017