MUTATIONAL SPECTROMETRY WITHOUT PHENOTYPIC SELECTION - HUMAN MITOCHONDRIAL-DNA

By first separating mutant from nonmutant DNA sequences on the basis o f their melting temperatures and then increasing the number of copies by high-fidelity DNA amplification, we have developed a method that al lows observation of point mutations in biological samples at fractions at or above 10(-6). Using this method, we have observed the hotspot p oint mutations that lie in 100 base pairs of the mitochondrial genome in samples of cultured cells and human tissues, To date, 19 mutants ha ve been isolated, their fractions ranging from 4 x 10(-4) down to the limit of detection, We performed specific tests to determine if the ob served signals were artefacts arising from contamination, polymerase e rrors during PCR or DNA adducts created during the procedure, We also tested the possibilities that DNA replication mismatch intermediates, or endogenous DNA adducts that were originally present in the cells, w ere included with true mutants in our separation Steps and converted t o mutants during PCR, We show that while most of the mutants behave as double-stranded point mutants in the cells, some appear to arise at l east in part from mismatch intermediates or cellular DNA adducts, This technology is therefore sufficient for the observation of the spectru m of point mutations' in human mitochondrial DNA and is a tool for dis covering the primary causes of these mutations.