Background: The phenotypic expression of disorders caused by point mutations, deletions or depletions within the mitochondrial genome (mtDNA) is heterogeneous. This relates to the phenomena of heteroplasmy, tissue threshold as well as the distribution of mutant DNA among tissues. Hence, the diagnostics of these disorders demands highly specific, sensitive and quantitative methods.
Methods: We have developed an allele-specific quantitative real-time PCR method for the detection of two of the most prevalent disease causing mitochondrial mutations, m.3243A>G (MELAS) and m.8993T>G (NARP). Locked Nucleic Acid (LNA) modified primers were used to obtain high allele specificity. In order to monitor mtDNA depletion a real-time method for mtDNA/nuclear DNA copy number ratio determination was developed.
Results: Rapid and sensitive detection and quantification of MELAS and NARP mtDNA alleles were achieved. Heteroplasmy levels as low as 0.01% could be detected, and the mtDNA/nuclear DNA ratio could be determined.
Conclusions: The present method that allows simultaneous determination of heteroplasmy levels and mtDNA/nuclear DNA copy number ratio, will provide a useful tool in molecular diagnostics and in future epidemiological studies of mitochondrial diseases.