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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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Target sequence in Trp53 locus used for RMC assay. A schematic drawing of the mouse Trp53 locus with the location of RMC target sequence is shown. Location of the TaqI restriction site is indicated, and control forward (Fwd C), control reverse (Rev C), mutant-specific forward (Fwd M) and mutant-specific reverse (Rev M) primers are designated with small arrows.
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Figure 4: Target sequence in Trp53 locus used for RMC assay. A schematic drawing of the mouse Trp53 locus with the location of RMC target sequence is shown. Location of the TaqI restriction site is indicated, and control forward (Fwd C), control reverse (Rev C), mutant-specific forward (Fwd M) and mutant-specific reverse (Rev M) primers are designated with small arrows.

Mentions: A TaqI restriction site located in intron IV of the Trp53 locus on chromosome 11 was used to develop the mouse RMC assay. The target site is illustrated in Figure 4. This site is located in an intronic region of no known function that is poorly conserved across species, suggesting that it is likely to be genetically neutral. Two pairs of primers from the Trp53 Intron IV target sequence were selected for use that have equivalent amplification efficiencies, one pair adjacent to the target TaqI site (control primers) and one pair flanking the TaqI site (mutant-specific primers). Total genomic DNA is extensively digested with TaqI restriction enzyme. The frequency of uncut wild-type TaqI sites that remain in the genomic DNA after digestion must be very low in order for rare mutations to be detectable. If the frequency of uncleaved, wild-type products captured is not in the same range as the frequency of mutant products, the vast majority of molecules captured and sequenced will be wild type making detection of mutants laborious.Figure 4.


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)

Target sequence in Trp53 locus used for RMC assay. A schematic drawing of the mouse Trp53 locus with the location of RMC target sequence is shown. Location of the TaqI restriction site is indicated, and control forward (Fwd C), control reverse (Rev C), mutant-specific forward (Fwd M) and mutant-specific reverse (Rev M) primers are designated with small arrows.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Target sequence in Trp53 locus used for RMC assay. A schematic drawing of the mouse Trp53 locus with the location of RMC target sequence is shown. Location of the TaqI restriction site is indicated, and control forward (Fwd C), control reverse (Rev C), mutant-specific forward (Fwd M) and mutant-specific reverse (Rev M) primers are designated with small arrows.
Mentions: A TaqI restriction site located in intron IV of the Trp53 locus on chromosome 11 was used to develop the mouse RMC assay. The target site is illustrated in Figure 4. This site is located in an intronic region of no known function that is poorly conserved across species, suggesting that it is likely to be genetically neutral. Two pairs of primers from the Trp53 Intron IV target sequence were selected for use that have equivalent amplification efficiencies, one pair adjacent to the target TaqI site (control primers) and one pair flanking the TaqI site (mutant-specific primers). Total genomic DNA is extensively digested with TaqI restriction enzyme. The frequency of uncut wild-type TaqI sites that remain in the genomic DNA after digestion must be very low in order for rare mutations to be detectable. If the frequency of uncleaved, wild-type products captured is not in the same range as the frequency of mutant products, the vast majority of molecules captured and sequenced will be wild type making detection of mutants laborious.Figure 4.

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