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1 Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899.
2 University of Maryland Biotechnology Institute, Center for Advanced Research in Biotechnology, Rockville, MD 20850.
3 Institute for Molecular and Human Genetics, Georgetown University Medical Center, Washington, DC 20007.
aAddress correspondence to this author at: 100 Bureau Dr., MS 8311, National Institute of Standards and Technology, Gaithersburg, MD 20899-8311. Fax 301-975-8505; e-mail barbara.levin{at}nist.gov.
Background: 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 wild-type sequence.
Conclusion: This methodology permits easy detection of low concentrations of the MELAS A3243G mutation in blood by standard PCR and sequencing methods.
The following articles in journals at HighWire Press have cited this article:
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 M. Urata, Y. Wada, S. H. Kim, W. Chumpia, Y. Kayamori, N. Hamasaki, and D. Kang High-Sensitivity Detection of the A3243G Mutation of Mitochondrial DNA by a Combination of Allele-Specific PCR and Peptide Nucleic Acid-Directed PCR Clamping Clin. Chem., November 1, 2004; 50(11): 2045 - 2051. [Abstract] [Full Text] [PDF]
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