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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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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.
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Figure 2: 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.

Mentions: High concentration TaqαI (100 000 U/ml; New England Biolabs catalog# R0149M) is used to maximize enzyme concentration in the reaction. TaqαI is a mutant form of the enzyme that has a two amino acid replacement at its amino terminus. This allows for a higher level of expression without interfering with its catalytic properties. Achieving sufficient digestion of genomic DNA with TaqI is a critical step in this protocol. The quality control testing provided by the supplier is designed to ensure sufficient digestion efficiency for analysis of digested DNA in bulk. For our application, where DNA molecules are analyzed individually, we have found that the quality of TaqαI can vary between lots. Small quantities of several lots of TaqαI enzyme should be tested first with one DNA sample and then the best lot then acquired in an adequate amount to complete the experiment (Figure 2).Figure 2.


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)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: 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.
Mentions: High concentration TaqαI (100 000 U/ml; New England Biolabs catalog# R0149M) is used to maximize enzyme concentration in the reaction. TaqαI is a mutant form of the enzyme that has a two amino acid replacement at its amino terminus. This allows for a higher level of expression without interfering with its catalytic properties. Achieving sufficient digestion of genomic DNA with TaqI is a critical step in this protocol. The quality control testing provided by the supplier is designed to ensure sufficient digestion efficiency for analysis of digested DNA in bulk. For our application, where DNA molecules are analyzed individually, we have found that the quality of TaqαI can vary between lots. Small quantities of several lots of TaqαI enzyme should be tested first with one DNA sample and then the best lot then acquired in an adequate amount to complete the experiment (Figure 2).Figure 2.

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