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  • Title: Quantitation of heteroplasmy of mitochondrial tRNA(Leu(UUR)) gene using PCR-SSCP.
    Author: Tanno Y, Yoneda M, Tanaka K, Tanaka H, Yamazaki M, Nishizawa M, Wakabayashi K, Ohama E, Tsuji S.
    Journal: Muscle Nerve; 1995 Dec; 18(12):1390-7. PubMed ID: 7477061.
    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.
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