Quantitation of heteroplasmy of mitochondrial trnaLeu(UUR) gene using PCR‐SSCP

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Abstract
We have devised a novel method for quantitative analysis of the MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes) tRNALeu(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 encephalo‐myopathies. We applied the method for quantitation of mutant mitochondrial DNA carrying a single base substitution in the tRNALeu(UUR) gene in two autopsied cases of MELAS. In both cases, the mutant mtDNA is abundantly present (82–95%) with little variation among tissues. © 1995 John Wiley & Sons, Inc.