title: Quantitation of heteroplasmy of mitochondrial tRNA(Leu(UUR)) gene using PCR-SSCP.
abstract: We have devised a novel method for quantitative analysis of the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) tRNA(Leu(UUR)) mutation of mitochondrial DNA using a PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) method, and compared the results obtained using the PCR-SSCP method with those obtained using other methods including Southern blotting, last one cycle hot PCR, and conventional PCR-RFLP (restriction fragment length polymorphism). The standard curve obtained using the PCR-SSCP method is linear, with a correlation coefficient of 0.999; it was determined that this method is more accurate than other methods for quantitative analysis. The PCR-SSCP method does not require restriction digestions, thereby avoiding potential problems of partial digestions or heteroduplex formation during PCR. The method is quite simple and should have a broad range of application for quantitation of mutant mtDNAs in various mitochondrial encephalomyopathies. We applied the method for quantitation of mutant mitochondrial DNA carrying a single base substitution in the tRNA(Leu(UUR)) gene in two autopsied cases of MELAS. In both cases, the mutant mtDNA is abundantly present (82-95%) withd little variation among tissues.
mesh_terms: D001483:Base Sequence; D015139:Blotting, Southern; D002648:Child; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004272:DNA, Mitochondrial / Q000235:genetics*; D005260:Female; D006801:Humans; D017241:MELAS Syndrome / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D018807:Polymorphism, Single-Stranded Conformational; D012356:RNA, Transfer, Leu / Q000235:genetics*
chemical_list: D004272:DNA, Mitochondrial; D012356:RNA, Transfer, Leu