Mutational spectrometry without phenotypic selection: human mitochondrial DNA

Abstract

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.

A sensitive scanning technology for low frequency nuclear point mutations in human genomic DNA

“…Genomic DNA was isolated from over 3 × 10 8 MNNG-treated and untreated MT1 cells without exposing the DNA to either phenol or anion exchange resins (8). The genomic DNA was digested with two 'inexpensive' endonucleases, HaeIII and XbaI (New England Biolabs, Beverly, MA), at 1 U enzyme/µg DNA and 2-3 mg DNA/ml overnight to liberate the target sequence embedded in a 482 bp fragment representing the APC cDNA bp 8422-8903.…”

“…The initial copy numbers of the APC target sequence and a 205 bp human mitochondrial DNA sequence (mitochondrial bp 10011-10215) (used as a reference sequence) in the restriction digestion were measured based on competitive PCR followed by CDCE separation (8). In brief, a small aliquot of the sample was mixed with known copies of an artificial mutant and subjected to PCR amplification (see below).…”

“…These efforts are built upon the original demonstration of Fischer and Lerman that small differences in DNA melting temperatures created clear and discernible differences in the electrophoretic mobility of DNA upon moving through a gel matrix under partially denaturing conditions (7). We have extended their approach to constant denaturant capillary electrophoresis (CDCE) which, in combination with high fidelity PCR, has enabled us to scan for point mutations in a 100 bp mitochondrial DNA sequence in human cells and tissues (8,9). Reproducible mitochondrial point mutational hot-spots were discovered at frequencies of from 10 -5 to greater than 10 -4 (9)(10)(11).…”

Analysis of mutational spectra by denaturing capillary electrophoresis

“…Mitochondrial mutations-In Khrapko et al 15,38,[55][56][57] , Coller et al 39 and Marcelino et al 58 the combination of high fidelity DNA amplification and constant denaturing capillary electrophoresis was used to discover the point mutations distributed in a 100 bp mitochondrial DNA sequence in human DNA replication by human DNA polymerase gamma, cells in culture, various tissues and derived tumors. Their work demonstrated for the first time that treatment of human cells with a chemical mutagen induced a reproducible set of mutations in mitochondrial DNA, that the spectrum of the "spontaneous" point mutations found in human cell cultures were not significantly different from those found in multiple human tissues and their derived tumors and that this in vivo mutational spectrum in the lung was not affected by cigarette smoking.…”

“…The same method of allele separation was first demonstrated on a commercial capillary DNA sequencing in 2001 73,108 . DCE have been applied to 7 different capillary DNA sequencing instrument, ranging from single capillary up to 384 capillaries 38,56,73,74,78,81,82,84,89,105 . Due to the large variety of instrument and instrument protocols and setting, detailed running descriptions will not be given for each instrument.…”