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Isolation of genes from complex sources of mammalian genomic DNA using exon amplification

Abstract

Modifications to exon amplification have been instituted that increase its speed, efficiency and reliability. Exons were isolated from target human or mouse genomic DNA sources ranging from 30 kilobases (kb) to 3 megabases (Mb) in complexity. The efficiency was dependent upon the amount of input DNA, and ranged from isolation of an exon for every 20 kb to an exon for every 80 kb of target genomic DNA. In these studies, several novel genes and a smaller number of genes isolated previously that reside on human chromosome 9 have been identified. These results indicate that exon amplification is presently adaptable to large scale isolation of exons from complex sources of genomic DNA.

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References

  1. Monaco, A.P. et al. Isolation of candidate cDNAs for portions of the Duchenne muscular dystrophy gene. Nature 323, 646–650 (1986).

    Article  CAS  PubMed  Google Scholar 

  2. Page, D.C. et al. The sex-determining region of the human Y chromosome encodes a finger protein. Cell 51, 1091–1104 (1987).

    Article  CAS  PubMed  Google Scholar 

  3. Rommens, J.M. et al. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 246, 1059–1065 (1989).

    Article  Google Scholar 

  4. Call, K.M. et al. Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor locus. Cell 60, 509–520 (1990).

    Article  CAS  PubMed  Google Scholar 

  5. Fearon, E.R. et al. Identification of a chromosome 18q gene that is altered in colorectal cancers. Science 247, 49–56 (1990).

    Article  CAS  PubMed  Google Scholar 

  6. Bird, A. CpG-rich islands and the function of DNA methylation. Nature 321, 209–213 (1986).

    Article  CAS  PubMed  Google Scholar 

  7. Liu, P., Legerski, R. & Siciliano, M.J. Isolation of human transcribed sequences from human-rodent somatic cell hybrids. Science 246, 813–815 (1989).

    Article  CAS  PubMed  Google Scholar 

  8. Corbo, L., Maley, J.A., Nelson, D.L. & Caskey, C.T. Direct cloning of human transcripts with hnRNA from hybrid cell lines. Science 249, 652–655 (1990).

    Article  CAS  PubMed  Google Scholar 

  9. Jones, K.W., Chevrette, M., Shapero, M.H. & Fournier, R.E.K. Generation of region-and species-specific expressed gene probes from somatic cell hybrids Nature Genet. 1, 278–283 (1992).

    Article  CAS  PubMed  Google Scholar 

  10. Lovett, M., Kere, J. & Hinton, L.M. Direct selection: A method for the isolation of cDNAs encoded by large genomic fragments. Proc. natn. Acad. Sci. U.S.A. 88, 9628–9632 (1991).

    Article  CAS  Google Scholar 

  11. Parimoo, S., Patanjali, S.R., Shulka, H., Chaplin, D.D. & Weissman, S.M. cDNA selection: Efficient PCR approach for the selection of cDNAs in large genomic DNA fragments. Proc. natn. Acad. Sci. U.S.A. 88, 9623–9627 (1991).

    Article  CAS  Google Scholar 

  12. Duyk, G.M., Kim, S., Myers, R.M. & Cox, D.R. Exon trapping: a genetic screen to identify candidate transcribed sequences in cloned mammalian genomic DNA. Proc. natn. Acad. Sci. U.S.A. 87, 8995–8999 (1990).

    Article  CAS  Google Scholar 

  13. Auch, D. & Reth, M. Exon trap cloning: using PCR to rapidly detect and clone exons from genomic DNA fragments. Nucl. Acids Res. 18, 6743–6744 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Buckler, A.J. et al. Exon amplification: a strategy to isolate mammalian genes based on RNA splicing. Proc. natn. Acad. Sci. U.S.A. 88, 4005–4009 (1991).

    Article  CAS  Google Scholar 

  15. Church, D.M. et al. Identification of human chromosome 9 specific genes using exon amplification, Hum. molec. Genet. 2, 1915–1920 (1993).

    Article  CAS  PubMed  Google Scholar 

  16. Vulpe, C., Levinson, B., Whitney, S., Packman, S. & Gitschier, J. Isolation of a candidate gene for Menkes disease and evidence that it encodes a copper-transporting ATPase. Nature Genet. 3, 7–13 (1993).

    Article  CAS  PubMed  Google Scholar 

  17. Trofatter, J.A. et al. A novel moesin-, ezrin-, radixin-llke gene is a candidate for the neurofibromatosis 2 tumor suppressor. Cell 72, 791–800 (1993).

    Article  CAS  PubMed  Google Scholar 

  18. Taylor, S.A.M. et al. Cloning of the α-adducin gene from the Huntington's disease candidate region of human chromosome 4 by exon amplification. Nature Genet. 1, 697–703 (1992).

    Google Scholar 

  19. Ambrose, C. et al. A novel G protein-coupled receptor kinase maps to the Huntington's disease candidate region. Hum. molec. Genet. 1, 697–703 (1992).

    Article  CAS  PubMed  Google Scholar 

  20. Duyao, M.P. et al. A gene from 4p16.3 with similarity to a superfamily of transporter proteins. Hum. molec. Genet. 2, 673–676 (1993).

    Article  CAS  PubMed  Google Scholar 

  21. The Huntington's Disease Collaborative Research Group. A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington's disease chromosomes. Cell 72, 971–983 (1993).

    Article  Google Scholar 

  22. Vidal, S.M., Malo, D., Vogan, K., Skamene, E. & Gros, P. Natural resistance to infection with intracellular parasites: Isolation of a candidate for Bcg. Cell 73, 469–85 (1993).

    Article  CAS  PubMed  Google Scholar 

  23. Mardon, H.J., Sebastio, G. & Barelle, F. A role for exon sequences in alternative splicing of the human fibronectin gene. Nucl. Acids Res. 15, 7725–7773 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Cooper, T.A. & Ordahl, C.P. Nucleotide substitutions within the cardiac troponin T alternative exon disrupt pre-mRNA alternative splicing. Nucl. Acids Res. 17, 7905–7921 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hodges, P.E. & Rosenberg, L.E. The spfash mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing. Proc. natn. Acad. Sci. U.S.A. 86, 4142–4146 (1989).

    Article  CAS  Google Scholar 

  26. Weil, D. et al. A base substitution in the exon of a collagen gene causes alternative splicing and generates a structurally abnormal polypeptide In a patient with Ehlers-Danlos syndrome type VII. EMBO J. 8, 1705–1710 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Akli, S. et al. A “G” to “A” mutation at position–1 of a 5′ splice site in a late infantile form of Tay-Sachs disease. J. biol. Chem. 265, 7324–7330 (1990).

    CAS  PubMed  Google Scholar 

  28. Vasan, N.S. et al. A mutation in the pro alpha 2(l) gene (COL1A2) for type I procollagen in Ehlers-Danlos syndrome type VII: evidence suggesting that skipping of exon 6 in RNA splicing may be a common cause of the phenotype. Am. J. hum. Genet. 48, 305–317 (1991).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Craig, S.P., Buckle, V.J., Lamouroux, A. & Mallet, J. Localization of the human dopamine beta hydroxylase (DBH) gene to chromosome 9q34. Cytogenet. Cell Genet. 48, 48–50 (1988).

    Article  CAS  PubMed  Google Scholar 

  30. Altschul, S.F., Gish, W., Miller, W., Myers, E. & Lippman, D.J. Basic local alignment search tool. J. molec. Biol. 215, 403–410 (1990).

    Article  CAS  PubMed  Google Scholar 

  31. Lindern, M.V. et al. The translocation (6;9), associated with a specific subtype of acute myeloid leukemia, results in the fusion of two genes, dek and can, and the expression of a chimeric, leukemia-specific dek-can mRNA. Molec. cell. Biol. 12, 1687–1697 (1992).

    Article  Google Scholar 

  32. Adams, B. et al. Pax-5 encodes the transcription factor BSAP and is expressed in B lymphocytes, the developing CNS, and adult testis. Genes Dev. 6, 158–1607 (1992).

    Article  Google Scholar 

  33. Stapleton, P., Weith, A., Urbanek, P., Kozmik, Z. & Busslinger, M. Chromosomal localization of seven PAX genes and cloning of a novel family member, PAX-9. Nature Genet. 3, 292–298 (1993).

    Article  CAS  PubMed  Google Scholar 

  34. Lidberg, U. et al. Genomic organization, sequence analysis, and chromosomal localization of the human carboxyl ester lipase (CEL) gene and a CEL-like (CELL) gene. Genomics 13, 630–640 (1992).

    Article  CAS  PubMed  Google Scholar 

  35. Williams, M.E. et al. Structure and functional expression of an omega-conotoxin-sensitive N-type calcium channel. Science 257, 389–395 (1992).

    Article  CAS  PubMed  Google Scholar 

  36. Adams, M.D. et al. Complementary DNA sequencing: expressed sequence tags and human genome project. Science 252, 1651–1656 (1992).

    Article  Google Scholar 

  37. Obar, R.A., Collins, C.A., Hammarback, J.A., Shpetner, H.S. & Vallee, R.B. Molecular cloning of the microtubule-associated mechanochemioal enzyme dynamin reveals homology with a new family of GTP-binding proteins. Nature 347, 256–261 (1990).

    Article  CAS  PubMed  Google Scholar 

  38. Takenaga, K., Nakamura, Y., Tokunaga, K., Kageyama, H. & Sakiyama, S. Isolation and characterization of a cDNA that encodes mouse fibroblast tropomyosin isoform 2. Molec. cell. Biol. 8, 5561–5565 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Hempel, W.M. & DeFranco, A.L. Expression of phospholipase C isozymes by murine B lymphocytes. J. Immunol. 146, 3713–3720 (1991).

    CAS  PubMed  Google Scholar 

  40. Bellefroid, E.J. et al. The human genome contains hundreds of genes coding for finger proteins of the kruppel type. DNA 8, 377–387 (1989).

    Article  CAS  PubMed  Google Scholar 

  41. Ayer, D.E., Kretzner, L. & Eisenman, R.N. Mad: a heterodimeric partner for max that antagonizes myc transcriptional activity. Cell 72, 211–222 (1993).

    Article  CAS  PubMed  Google Scholar 

  42. Kwiatkowski, D.J. et al. Report and abstracts of the Second International Workshop on Human Chromosome 9 Mapping 1993. Cytogenet. Cell Genet. 64, 93–121 (1993).

    Article  CAS  PubMed  Google Scholar 

  43. Henikoff, S., Keene, M., Fechtel, K. & Fristrom, J. Gene within agene: nested Drosophila genes encode unrelated proteins on opposite strands. Cell 44, 33–42 (1986).

    Article  CAS  PubMed  Google Scholar 

  44. Williams, T. & Fried, M. A mouse locus at which transcription from both DNA strands produces mRNs complementary at their 3′ ends.Nature 322, 275–277 (1986).

    Article  CAS  PubMed  Google Scholar 

  45. Spencer, C., Gietz, D. & Hodgetts, R. Overlapping transcription units in the DOPA decarboxylase region of Drosophila. Nature 322, 279–281 (1986).

    Article  CAS  PubMed  Google Scholar 

  46. Adelman, J.P., Bond, C.T., Douglass, J. & Herbert, E. Two mammalian genes transcribed from opposite strands of the same locus. Science 235, 1514–1517 (1987).

    Article  CAS  PubMed  Google Scholar 

  47. Duin, M.V. et al. Conserved pattern of antisense overlapping transcription in the homologous human ERCC-1 and yeast RAD10 DNA repair gene regions. Molec. cell. Biol. 9, 1794–1798 (1989).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Okubo, K. et al. Large scale cDNA sequencing for analysis of quantitative and qualitative aspects of gene expression. Nature Genet. 2, 173–179 (1992).

    Article  CAS  PubMed  Google Scholar 

  49. Khan, A.S. et al. Single pass sequencing and physical and genetic mapping of human brain cDNAs. Nature Genet. 2, 180–185 (1992).

    Article  CAS  PubMed  Google Scholar 

  50. Warburton, D. et al. Monochromosomal rodent-human hybrids from microcell fusion of human lymphoblastoid cells containing an inserted dominant selectable marker. Genomics 6, 358–366 (1990).

    Article  CAS  PubMed  Google Scholar 

  51. Sanger, F., Nicklen, S. & Coulson, A.R. DNA sequencing with chain terminating inhibitors. Proc. natn. Acad. Sci. U.S.A. 74, 5463–5467 (1977).

    Article  CAS  Google Scholar 

  52. Innis, M.A., Myambo, K.B., Gelfand, D.H. & Brow, M.A.D. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. natn. Acad. Sci. U.S.A. 85, 9436–9440 (1988).

    Article  CAS  Google Scholar 

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Church, D., Stotler, C., Rutter, J. et al. Isolation of genes from complex sources of mammalian genomic DNA using exon amplification. Nat Genet 6, 98–105 (1994). https://doi.org/10.1038/ng0194-98

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