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        Scanning Low-Frequency Point Mutants in the Mitochondrial Genome Using Constant Denaturant Capillary Electrophoresis

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        The ability to measure rare mutational events in mitochondrial genomes from human blood and tissues without resorting to phenotypic selection is invaluable. It is essential for the study of the cause(s) of mitochondrial mutation and for the identification of mutations associated with diseases. We have developed a highly sensitive technique termed constant denaturant capillary electrophoresis (CDCE) (1 ), permitting measurement of mitochondrial point mutations at fractions as low as 106 in human cells and tissues (2 ). CDCE is based on the earlier development of denaturing gradient gel electrophoresis (DGGE) by Fischer and Lerman (3 ) and constant denaturing gradient gel electrophoresis (CDGE) by Hovig (4 ). When a DNA consists of two contiguous “isomelting domains” with different melting temperatures, under partially denaturing condition it alternates in rapid equilibrium between the double helix state and the partially melted state. The later form has an Y-shaped configuration and, thus, lower mobility through the gel matrix than the double helix. The distribution of the “fast” and “slow” states governs the velocity under electrophoretic conditions. Base changes in the low-melting domain result in changes in the melting behavior and, hence, the mobility of the sequence under the denaturing condition of electrophoresis, allowing variants to be separated from each other. Unlike DGGE and CDGE, CDCE employs replaceable linear (uncrosslinked) polyacrylamide and capillary electrophoresis (1 ). A zone of elevated temperature along the capillary maintains the constant denaturing condition necessary for DNA separation. The result is fast separation, improved resolution, and higher sensitivity (1 ).
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