title: A random mutation capture assay to detect genomic point mutations in mouse tissue.
abstract: Herein, a detailed protocol for a random mutation capture (RMC) assay to measure nuclear point mutation frequency in mouse tissue is described. This protocol is a simplified version of the original method developed for human tissue that is easier to perform, yet retains a high sensitivity of detection. In contrast to assays relying on phenotypic selection of reporter genes in transgenic mice, the RMC assay allows direct detection of mutations in endogenous genes in any mouse strain. Measuring mutation frequency within an intron of a transcribed gene, we show this assay to be highly reproducible. We analyzed mutation frequencies from the liver tissue of animals with a mutation within the intrinsic exonuclease domains of the two major DNA polymerases, δ and ε. These mice exhibited significantly higher mutation frequencies than did wild-type animals. A comparison with a previous analysis of these genotypes in Big Blue mice revealed the RMC assay to be more sensitive than the Big Blue assay for this application. As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible. This assay provides a versatile alternative to transgenic mouse models for the study of mutagenesis in vivo.
authors: Department of Pathology, University of Washington and Department of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. jhw5@uw.edu
authors: Jocelyn H
authors: JH
authors: Wright
authors: Kristina L
authors: KL
authors: Modjeski
authors: Jason H
authors: JH
authors: Bielas
authors: Bradley D
authors: BD
authors: Preston
authors: Nelson
authors: N
authors: Fausto
authors: Lawrence A
authors: LA
authors: Loeb
authors: Jean S
authors: JS
authors: Campbell
mesh_terms: D000818:Animals; D004252:DNA Mutational Analysis; D004257:DNA Polymerase II / Q000235:genetics; D004258:DNA Polymerase III / Q000235:genetics; D016678:Genome; D051379:Mice; D008810:Mice, Inbred C57BL; D016296:Mutagenesis; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results
chemical_list: D004257:DNA Polymerase II; D004258:DNA Polymerase III