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. 1996 Mar 15;24(6):1105–1111. doi: 10.1093/nar/24.6.1105

Scanning for genes in large genomic regions: cosmid-based exon trapping of multiple exons in a single product.

N A Datson 1, E van de Vosse 1, H G Dauwerse 1, M Bout 1, G J van Ommen 1, J T den Dunnen 1
PMCID: PMC145763  PMID: 8604345

Abstract

To facilitate the scanning of large genomic regions for the presence of exonic gene segments we have constructed a cosmid-based exon trap vector. The vector serves a dual purpose since it is also suitable for contig construction and physical mapping. The exon trap cassette of vector sCOGH1 consists of the human growth hormone gene driven by the mouse mettallothionein-1 promoter. Inserts are cloned in the multicloning site located in intron 2 of the hGH gene. The efficiency of the system is demonstrated with cosmids containing multiple exons of the Duchenne Muscular Dystrophy gene. All exons present in the inserts were successfully retrieved and no cryptic products were detected. Up to seven exons were isolated simultaneously in a single spliced product. The system has been extended by a transcription-translation-test protocol to determine the presence of large open reading frames in the trapped products, using a combination of tailed PCR primers directing protein synthesis in three different reading frames, followed by in vitro transcription-translation. Having larger stretches of coding sequence in a single exon trap product rather than small single exons greatly facilitates further analysis of potential genes and offers new possibilities for direct mutation analysis of exon trap material.

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Selected References

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  1. Ahn A. H., Kunkel L. M. The structural and functional diversity of dystrophin. Nat Genet. 1993 Apr;3(4):283–291. doi: 10.1038/ng0493-283. [DOI] [PubMed] [Google Scholar]
  2. Andreadis A., Nisson P. E., Kosik K. S., Watkins P. C. The exon trapping assay partly discriminates against alternatively spliced exons. Nucleic Acids Res. 1993 May 11;21(9):2217–2221. doi: 10.1093/nar/21.9.2217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Auch D., Reth M. Exon trap cloning: using PCR to rapidly detect and clone exons from genomic DNA fragments. Nucleic Acids Res. 1990 Nov 25;18(22):6743–6744. doi: 10.1093/nar/18.22.6743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Buckler A. J., Chang D. D., Graw S. L., Brook J. D., Haber D. A., Sharp P. A., Housman D. E. Exon amplification: a strategy to isolate mammalian genes based on RNA splicing. Proc Natl Acad Sci U S A. 1991 May 1;88(9):4005–4009. doi: 10.1073/pnas.88.9.4005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Church D. M., Stotler C. J., Rutter J. L., Murrell J. R., Trofatter J. A., Buckler A. J. Isolation of genes from complex sources of mammalian genomic DNA using exon amplification. Nat Genet. 1994 Jan;6(1):98–105. doi: 10.1038/ng0194-98. [DOI] [PubMed] [Google Scholar]
  6. Datson N. A., Duyk G. M., Van Ommen J. B., Den Dunnen J. T. Specific isolation of 3'-terminal exons of human genes by exon trapping. Nucleic Acids Res. 1994 Oct 11;22(20):4148–4153. doi: 10.1093/nar/22.20.4148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Den Dunnen J. T., Grootscholten P. M., Dauwerse J. G., Walker A. P., Monaco A. P., Butler R., Anand R., Coffey A. J., Bentley D. R., Steensma H. Y. Reconstruction of the 2.4 Mb human DMD-gene by homologous YAC recombination. Hum Mol Genet. 1992 Apr;1(1):19–28. doi: 10.1093/hmg/1.1.19. [DOI] [PubMed] [Google Scholar]
  8. Dietz H. C., Kendzior R. J., Jr Maintenance of an open reading frame as an additional level of scrutiny during splice site selection. Nat Genet. 1994 Oct;8(2):183–188. doi: 10.1038/ng1094-183. [DOI] [PubMed] [Google Scholar]
  9. Duyk G. M., Kim S. W., Myers R. M., Cox D. R. Exon trapping: a genetic screen to identify candidate transcribed sequences in cloned mammalian genomic DNA. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8995–8999. doi: 10.1073/pnas.87.22.8995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Evans G. A., Lewis K., Rothenberg B. E. High efficiency vectors for cosmid microcloning and genomic analysis. Gene. 1989 Jun 30;79(1):9–20. doi: 10.1016/0378-1119(89)90088-7. [DOI] [PubMed] [Google Scholar]
  11. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Krizman D. B., Berget S. M. Efficient selection of 3'-terminal exons from vertebrate DNA. Nucleic Acids Res. 1993 Nov 11;21(22):5198–5202. doi: 10.1093/nar/21.22.5198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Petrij F., Giles R. H., Dauwerse H. G., Saris J. J., Hennekam R. C., Masuno M., Tommerup N., van Ommen G. J., Goodman R. H., Peters D. J. Rubinstein-Taybi syndrome caused by mutations in the transcriptional co-activator CBP. Nature. 1995 Jul 27;376(6538):348–351. doi: 10.1038/376348a0. [DOI] [PubMed] [Google Scholar]
  14. Roest P. A., Roberts R. G., Sugino S., van Ommen G. J., den Dunnen J. T. Protein truncation test (PTT) for rapid detection of translation-terminating mutations. Hum Mol Genet. 1993 Oct;2(10):1719–1721. doi: 10.1093/hmg/2.10.1719. [DOI] [PubMed] [Google Scholar]
  15. Searle P. F., Stuart G. W., Palmiter R. D. Building a metal-responsive promoter with synthetic regulatory elements. Mol Cell Biol. 1985 Jun;5(6):1480–1489. doi: 10.1128/mcb.5.6.1480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Selden R. F., Howie K. B., Rowe M. E., Goodman H. M., Moore D. D. Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol Cell Biol. 1986 Sep;6(9):3173–3179. doi: 10.1128/mcb.6.9.3173. [DOI] [PMC free article] [PubMed] [Google Scholar]

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