Limits...
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.

Show MeSH

Related in: MedlinePlus

RMC assay: pictorial illustration of experimental steps. Photograph of a normal mouse liver is shown. Individual steps in the protocol are indicated with boxed numbers. In step 2, target sequences in the digested DNA are color coded with blue for wild type and red for mutant. Steps 3, 4 and 5 illustrate a typical experimental design using 96-well qPCR plates. The dilution factors suggested to test are shown in powers of 4 in the wells. In steps 3 and 5, the DNA standard curve is shown in gray, and analysis of six different DNA samples are shown in different colors. In step 4, wells marked ‘Blk’ are blanks to test for DNA contamination during the mutant screening step. Numbered experimental steps shown relate to numbers in the experimental flow chart in Figure 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113589&req=5

Figure 3: RMC assay: pictorial illustration of experimental steps. Photograph of a normal mouse liver is shown. Individual steps in the protocol are indicated with boxed numbers. In step 2, target sequences in the digested DNA are color coded with blue for wild type and red for mutant. Steps 3, 4 and 5 illustrate a typical experimental design using 96-well qPCR plates. The dilution factors suggested to test are shown in powers of 4 in the wells. In steps 3 and 5, the DNA standard curve is shown in gray, and analysis of six different DNA samples are shown in different colors. In step 4, wells marked ‘Blk’ are blanks to test for DNA contamination during the mutant screening step. Numbered experimental steps shown relate to numbers in the experimental flow chart in Figure 2.

Mentions: Flow diagram of RMC protocol. The sequence of experimental steps is shown with important considerations for each of those steps shown on the left. Boxed numbers refer to the same numbers step illustrated in Figure 3. A troubleshooting guide for this protocol is available in Supplementary Table I.


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)

RMC assay: pictorial illustration of experimental steps. Photograph of a normal mouse liver is shown. Individual steps in the protocol are indicated with boxed numbers. In step 2, target sequences in the digested DNA are color coded with blue for wild type and red for mutant. Steps 3, 4 and 5 illustrate a typical experimental design using 96-well qPCR plates. The dilution factors suggested to test are shown in powers of 4 in the wells. In steps 3 and 5, the DNA standard curve is shown in gray, and analysis of six different DNA samples are shown in different colors. In step 4, wells marked ‘Blk’ are blanks to test for DNA contamination during the mutant screening step. Numbered experimental steps shown relate to numbers in the experimental flow chart in Figure 2.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: RMC assay: pictorial illustration of experimental steps. Photograph of a normal mouse liver is shown. Individual steps in the protocol are indicated with boxed numbers. In step 2, target sequences in the digested DNA are color coded with blue for wild type and red for mutant. Steps 3, 4 and 5 illustrate a typical experimental design using 96-well qPCR plates. The dilution factors suggested to test are shown in powers of 4 in the wells. In steps 3 and 5, the DNA standard curve is shown in gray, and analysis of six different DNA samples are shown in different colors. In step 4, wells marked ‘Blk’ are blanks to test for DNA contamination during the mutant screening step. Numbered experimental steps shown relate to numbers in the experimental flow chart in Figure 2.
Mentions: Flow diagram of RMC protocol. The sequence of experimental steps is shown with important considerations for each of those steps shown on the left. Boxed numbers refer to the same numbers step illustrated in Figure 3. A troubleshooting guide for this protocol is available in Supplementary Table I.

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