Random Mutation Detection

Previous mutational assays able to identify rare random spontaneous mutations have ultimately been restricted to model systems. Although tissue culture and transgenic animal systems are powerful tools for identifying potential mutagens, they cannot accurately predict mutagenesis in humans. To permit the measurement of rare random mutation in human tissues, we developed the Random Mutation Capture (RMC) assay. The RMC assay is >100-fold more sensitive than previous methods that employ genomic selection, permits analysis of a large number of nucleotides, and can identify one mutant base pair among 109 wild-type base pairs.

It was with the development of this new technology that we were first able to provide the most convincing evidence to date for existence of a mutator phenotype in human cancers, a hypothesis proposed more than 30 years prior.

Although this assay was initially developed to study point mutation accumulation in the nuclear genome, we have since adapted it to resolve mitochondrial mutations and increased its resolution and throughput by “digitizing” the assay to more sensitively monitor base substitution and deletion mutations (Figure 1). This has allowed us to redefine the relationship among mitochondrial mutagenesis, cancer and aging.

For example, we recently demonstrated two surprising phenomena: 1) far fewer mitochondrial mutations arise in tumors than in normal healthy tissue, and 2) mitochondrial DNA exhibits mutagenic resistance to DNA-damaging agents.

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