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A random mutation capture assay to detect genomic point mutations in mouse tissue.

Wright JH, Modjeski KL, Bielas JH, Preston BD, Fausto N, Loeb LA, Campbell JS - Nucleic Acids Res. (2011)

Bottom Line: 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.As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington and Department of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. jhw5@uw.edu

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.

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The reproducibility of data from two RMC analyses of 9-month-old wild-type C57Bl/6 mice. Results from two different experiments performed on the same DNA samples are presented. Results from experiment 1 are shown in black, and those from experiment 2 are shown in gray. Each bar represents mutation frequencies calculated for individual mice. Mouse ‘code’ names are indicated below each pair of data bars.
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Figure 5: The reproducibility of data from two RMC analyses of 9-month-old wild-type C57Bl/6 mice. Results from two different experiments performed on the same DNA samples are presented. Results from experiment 1 are shown in black, and those from experiment 2 are shown in gray. Each bar represents mutation frequencies calculated for individual mice. Mouse ‘code’ names are indicated below each pair of data bars.

Mentions: We analyzed a cohort of 9-month-old, wild-type C57BL/6 mice using the RMC assay. Mice were sacrificed, and the left lobe of each mouse liver was harvested and quickly frozen. Genomic DNA was isolated from tissue pieces between 200 and 400 mg in weight. In independent experiments performed 6 months apart, the same DNA samples were digested with two different lots of TaqαI enzyme, and analyzed with different batches of qPCR reagents. Roughly one-fourth of a liver was homogenized to generate each DNA sample, and about 1/500 of that DNA sample was analyzed in each of the two independent experiments. Figure 5 shows a comparison of the mutation frequencies calculated for the mice from the two different experiments. Each bar represents the mutation frequency calculated for the individual mice.Figure 5.


A random mutation capture assay to detect genomic point mutations in mouse tissue.

Wright JH, Modjeski KL, Bielas JH, Preston BD, Fausto N, Loeb LA, Campbell JS - Nucleic Acids Res. (2011)

The reproducibility of data from two RMC analyses of 9-month-old wild-type C57Bl/6 mice. Results from two different experiments performed on the same DNA samples are presented. Results from experiment 1 are shown in black, and those from experiment 2 are shown in gray. Each bar represents mutation frequencies calculated for individual mice. Mouse ‘code’ names are indicated below each pair of data bars.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113589&req=5

Figure 5: The reproducibility of data from two RMC analyses of 9-month-old wild-type C57Bl/6 mice. Results from two different experiments performed on the same DNA samples are presented. Results from experiment 1 are shown in black, and those from experiment 2 are shown in gray. Each bar represents mutation frequencies calculated for individual mice. Mouse ‘code’ names are indicated below each pair of data bars.
Mentions: We analyzed a cohort of 9-month-old, wild-type C57BL/6 mice using the RMC assay. Mice were sacrificed, and the left lobe of each mouse liver was harvested and quickly frozen. Genomic DNA was isolated from tissue pieces between 200 and 400 mg in weight. In independent experiments performed 6 months apart, the same DNA samples were digested with two different lots of TaqαI enzyme, and analyzed with different batches of qPCR reagents. Roughly one-fourth of a liver was homogenized to generate each DNA sample, and about 1/500 of that DNA sample was analyzed in each of the two independent experiments. Figure 5 shows a comparison of the mutation frequencies calculated for the mice from the two different experiments. Each bar represents the mutation frequency calculated for the individual mice.Figure 5.

Bottom Line: 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.As RMC does not require analysis of a particular gene, simultaneous analysis of mutation frequency at multiple genetic loci is feasible.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Washington and Department of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. jhw5@uw.edu

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.

Show MeSH
Related in: MedlinePlus