Nucleic Acids Research, Vol 25, Issue 4 685-693, Copyright © 1997 by Oxford University Press
K Khrapko, H Coller, P Andre, XC Li, F Foret, A Belenky, BL Karger and WG Thilly
By first separating mutant from nonmutant DNA sequences on the basis of
their melting temperatures and then increasing the number of copies by
high-fidelity DNA amplification, we have developed a method that allows
observation of point mutations in biological samples at fractions at or
above 10-6. Using this method, we have observed the hotspot point mutations
that lie in 100 base pairs of the mitochondrial genome in samples of
cultured cells and human tissues. To date, 19 mutants have been isolated,
their fractions ranging from 4x10-4 down to the limit of detection. We
performed specific tests to determine if the observed signals were
artefacts arising from contamination, polymerase errors 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, were included with true mutants in our
separation steps and converted to 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 least in part from mismatch intermediates or
cellular DNA adducts. This technology is therefore sufficient for the
observation of the spectrum of point mutations in human mitochondrial DNA
and is a tool for discovering the primary causes of these mutations.
ARTICLES
Mutational spectrometry without phenotypic selection: human mitochondrial DNA
Division of Toxicology, Center for Environmental Health Sciences, MIT, Cambridge, MA 02139, USA.
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