QUANTITATION OF HETEROPLASMY OF MITOCHONDRIAL TRNA(LEU(UUR)) GENE USING PCR-SSCP

We have devised a novel method for quantitative analysis of the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-l ike episodes) tRNA(Leu(UUR)) mutation of mitochondrial DNA using a PCR -SSCP (polymerase chain reaction-single-strand conformation polymorphi sm) method, and compared the results obtained using the PCR-SSCP metho d with those obtained using other methods including Southern blotting, last one cycle hot PCR, and conventional PCR-RFLP (restriction fragme nt length polymorphism). The standard curve obtained using the PCR-SSC P method is linear, with a correlation coefficient of 0.999; it was de termined that this method is more accurate than other methods for quan titative analysis, The PCR-SSCP method does not require restriction di gestions, thereby avoiding potential problems of partial digestions or heteroduplex formation during PCR, The method is quite simple and sho uld have a broad range of application for quantitation of mutant mtDNA s in various mitochondrial encephalomyopathies. We applied the method for quantitation of mutant mitochondrial DNA carrying a single base su bstitution in the tRNA(Leu(UUR)) gene in two autopsied cases of MELAS, In both cases, the mutant mtDNA is abundantly present (82-95%) with l ittle variation among tissues. (C) 1995 John Wiley & Sons, Inc.