Abstract
We have used the new high-throughput mutation-scanning technique temperature-gradient capillary electrophoresis (TGCE) for the identification of point mutations induced by N-ethyl-N-nitrosourea (ENU) in the mouse genome. TGCE detects the presence of heteroduplex molecules formed between a wild-type gene segment and the corresponding homologous segment containing an induced mutation or a naturally occurring single nucleotide polymorphism (SNP). Partially denatured heteroduplex molecules are resolved from homoduplexes by virtue of their differential mobilities during capillary electrophoresis conducted in a finely controlled temperature gradient. Simultaneous heteroduplex analysis of 96 amplicons ranging from 150 to 600 bp in size is achieved in approximately 45 min without the need for predetermining the melting profile of each fragment. Initially, we exploited known mouse mutations to develop TGCE protocols for analyzing unpurified PCR samples amplified from crude tail-DNA preparations. TGCE was then applied to the rapid identification of three new ENU-induced mutations recovered from regional mutagenesis screens of a segment of mouse Chromosome 7. Enzyme assays and quantitative reverse transcription-PCR (qRT-PCR) methods validated these new mutations. Our data demonstrate that rapid mutation scanning with TGCE, followed by sequence verification only of detected positives, is an efficient approach to the identification of point mutations in the mouse genome.
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References
Andrulis IL, Anton-Culver H, Beck J, Bove B, Boyd J, et al. (2002) Comparison of DNA- and RNA-based methods for detection of truncating BRCA1 mutations. Hum Mutat 20: 65–73
Aponte JL, Sega GA, Hauser LJ, Dhar MS, Withrow CM, et al. (2001) Point mutations in the murine fumarylacetoacetate gene: animal models for the human genetic disorder hereditary tyrosinemia type I. Proc Natl Acad Sci USA 98: 641–645
Balling R (2001) ENU mutagenesis: analyzing gene function in mice. Annu Rev Genomics Hum Genet 2: 463–492
Bedell MA, Largaespada DA, Jenkins NA, Copeland NG (1997) Mouse models of human disease. Part II. Recent progress and future directions. Genes Dev 11: 11–43
Boutin P, Vasseur F, Samson C, Wahl A, Frougel P (2001) Routine mutation screening of HNF-1alpha and GCK genes in MODY diagnosis: how effective are the techniques of dhplc and direct sequencing? Diabetologia 44: 775–778
Chen Y, Yee D, Dains K, Chatterjee A, Cavalcoli J, et al. (2000) Genotype-based screen for ENU-induced mutations in mouse embryonic stem cells. Nat Genet 24: 314–317
Coghill EL, Hugill A, Parkinson N, Davison C, Glenister P, et al. (2002) A gene-driven approach to the identification of ENU mutants in the mouse. Nat Genet 30: 255–256
Fernandes RJ, Skiena SS (2002) Microarray synthesis through multiple-use PCR primer design. Bioinformatics 1: S128–135
Gao Q, Yeung ES (2000) High-throughput detection of unknown mutations by using multiplexed capillary electrophoresis with poly(vinylpyrollidone) solution. Anal Chem 72: 2499–2506
Gelfi C, Vigano A, Palma SD, Righetti PG, Righetti SC, et al. (2002) Single–strand conformation polymorphism for p53 mutation by a combination of neutral pH buffer and temperature gradient in capillary electrophoresis. Electrophoresis 10: 1517–1523
Hrabe de Angelis M, Strivens M (2001) Large-scale production of mouse phenotypes: The search for animal models for inherited diseases in humans. Brief Bioinform 2: 170–180
Hrabe de Angelis MH, Flaswinkel H, Fuchs H, Rathkolb B, Soewarto D, et al (2000) Genome-wide, large scale production of mutant mice by ENU–mutagenesis. Nat Genet 25: 444–447
Huang JH, Liao WS (1994) Induction of the serum amyloid A3 gene by cytokines requires both C/EBP family proteins and a novel constitutive nuclear factor. Mol Cell Biol 14: 4475–4484
Jansen JG, van Teijlingen CM, Mohn GR, van Zeeland AA, Vrieling H (1994) AT basepairs are the main target for mutations at the Hprt locus of rat skin fibroblasts exposed in vitro to the monofunctional alkylating agent N-ethyl-N-nitrosourea. Mutagenesis 9: 417–421
Justice MJ (2000) Capitalizing on large-scale mouse mutagenesis screens. Nat Rev Genet 1: 109–115
Kampke T, Kieninger M, Mecklenburg M (2001) Efficient primer design algorithms. Bioinformatics 17: 214–225
Klebig ML, Wall MD, Potter MD, Rowe EL, Carpenter DA, et al. (2003) Mutations in the clathrin-assembly gene Picalm are responsible for the hematopoietic and iron metabolism abnormalities in fit1 mice. Proc Natl Acad Sci U S A 100: 8360–8365
Klein B, Weirich G, Brauch H (2001) DHPLC-based germline mutation screening in the analysis of the VHL tumor suppressor gene:usefulness and limitations. Hum Genet 108: 376–384
Li Q, Liu Z, Monroe M, Culiat CT (2002) Integrated platform for detection of DNA sequence variants using capillary electrophoresis. Electrophoresis 23: 1499–1511
Meek RL, Eriksen N, Benditt EP (1992) Murine serum amyloid A3 is a high density apolipoprotein and is secreted by macrophages. Proc Natl Acad Sci U S A 89: 7949–7952
MeislerMH (1996) The role of the laboratory mouse in the Human Genome Project. Am J Hum Genet 59: 764–771
Moldin SO, Farmer ME, Chin HR, Battey Jr JF (2001) Trans-NIH neuroscience initiatives on mouse phenotyping and mutagenesis. Mamm Genome 12: 575–581
Munroe RJ, Bergstrom RA, Zheng QY, Libby B, Smith R, et al. (2000) Mouse mutants from chemically mutagenized embryonic stem cells. Nat Genet 24: 318–321
Nolan PM, Peters J, Strivens M, Rogers D, Hagan J, et al. (2000) A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse. Nat Genet 25: 440–443
Pretsch W, Chatterjee B, Favor J, Merkle S, Sandulache R (1998) Molecular, genetic and biochemical characterization of lactate dehydrogenase-A enzyme activity mutations in Mus musculus. Mamm Genome 9: 144–149
Quwailid MM, Hugill A, Dear N, Vizor L, Wells S, et al. (2004) A gene-driven ENU-based approach to generating an allelic series in any gene. Mamm Genome 15: 585–591
Rastan S, Hough T, Kierman A, Hardisty R, Erven A, et al. (2004) Towards a mutant map of the mouse—new models of neurologic, behavioral, deafness, bone, renal and blood disorders. Genetica 122: 47–49
Rathkolb B, Fuchs E, Kold HJ, Renner-Muller I, Krebs O, et al. (2000) Large-scale N-ethyl-N-nitrosourea mutagenesis of mice—from phenotypes to genes. Exp Physiol 85: 635–644
Ravnik-Glavak M, Atkinson A, Glavac D, Dean M (2002) DHPLC screening of cystic fibrosis gene mutations. Hum Mutat 19: 374–383
Read JA, Winter VJ, Eszes CM, Sessions RB, Brady RL (2001) Structural basis for altered activity of M- and H-isozyme forms of human lactate dehydrogenase. Proteins 43: 175–185
Ribas G, Neville MJ, Campbell RD (2001) Single-nucleotide polymorphism detection by denaturing high-performance liquid chromatography and direct sequencing in genes in the MHC class III region encoding novel cell surface molecules. Immunogenetics 53: 369–381
Rinchik EM (1991) Chemical mutagenesis and fine-structure functional analysis of the mouse genome. Trends Genet 7: 15–21
Rinchik EM, Carpenter D, Johnson DK (2002) Functional annotation of mammalian genomic DNA sequence by high-efficiency mutagenesis: a fine-structure genetic mutation map of a 1- to 2-cM segment of mouse chromosome 7 corresponding to human chromosome 11p14-p15. Proc Natl Acad Sci U S A 99: 844–849
Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds.) Bioinformatics methods and protocols: Methods in molecular biology, (Totowa, NJ: Humana Press), pp 365–386
Russell WL, Kelly PR, Hunsicker PR, Bangham JW, Maddux SC (1979) Specific locus test shows ethylnitrosourea to be the most potent mutagen in the mouse. Proc Natl Acad Sci U S A 76: 5918–5922
Soewarto D, Fella C, Teubner A, Rathkolb B, Pargent W, et al. (2000) The large-scale Munich ENU-mouse-mutagenesis screen. Mamm Genome 11: 507–510
Yan H, Kinzler KW, Vogelstein B (2000) Genetic testing—present and future. Science 289: 1890–1892
Acknowledgment
We thank Frank Larimer, Tse Yuan Lu, and Melissa York for technical assistance in sequencing and initial set-up of TGCE. Edward Michaud contributed valuable comments in the preparation of this manuscript. Zhiyong Guo provided support with the Revelation™ mutation analysis software and Yesim Aydin-Son assisted with the qRTPCR assays. This work was funded by the Office of Biological and Environmental Research in the Department of Energy under the contract DE-AC05-00OR22725.
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This article has been authored by a contractor of the U.S. Government under contract DEAC05-00OR22725. Accordingly, the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725.
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Culiat, C.T., Klebig, M.L., Liu, Z. et al. Identification of mutations from phenotype-driven ENU mutagenesis in mouse Chromosome 7. Mamm Genome 16, 555–566 (2005). https://doi.org/10.1007/s00335-005-0032-0
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DOI: https://doi.org/10.1007/s00335-005-0032-0