S1 Nuclease Analysis and Direct Sequencing of Deleted Mitochondrial DNA in Myopathic Patients: Role of Directly Repeated Sequences in Deletion
Heteroplasmy of the normal-sized and the deleted mitochondrial DNA (mtDNA) has been observed in mitochondrial myopathy. To investigate the mechanism of mtDNA deletion, we analyzed the crossover sequences of mtDNA in the skeletal muscles of five patients with mitochondrial myopathy. We localized the deleted region using the combination of polymerase chain reaction and S1 nuclease digestion. Then, we directly sequenced the crossover regions of the deleted mitochondrial DNA without cloning. In Patient 1, a 7-bp directly repeated sequence of 5′-ATCCCCA-3′ was found at the boundaries of deleted segment spanning 7,039 bp between the ATPase 6 and the cytochrome b genes. In Patients 2, a 13-bp sequence of 5′-ACCTCCCTC ACCA-3′ was found in the boundaries of deleted segment spanning 4,977 bp between the ATPase 8 and the ND5 genes. In Patient 3, a 3-bp sequence of 5′-CCT-3′ was found in the boundaries of deleted segment spanning 3,717 bp between the ATPase 6 and the ND5 genes. In Patients 4 and 5 with multiple deletions, one of the mutant mitochondrial DNA was amplified. Direct sequencing of the mutant mtDNA revealed the same directly repeated sequence as in Patient 2. These directly repeated sequences may contribute to mitochondrial DNA deletions in human degenerative diseases.
KeywordsPolymerase Chain Reaction Method Mitochondrial Myopathy ATPase Subunit Nuclease Digestion Heteroduplex Formation
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