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PCR in Bioanalysis pp 127–144Cite as

In Vitro Transcription/Translation Analysis for the Identification of Translation-Terminating Mutations

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Part of the book series: Methods In Molecular Medicine™ ((MIMB,volume 92))

Abstract

The identification of mutations is very important in such aspects of molecular biology as medical diagnostics, ascertaining structure/function relationships, population genetic studies, and in confirming the authenticity of new candidate genes. Presently there are a variety of different techniques used to identify somatic and germline mutations. In some instances, gross genetic alterations are best characterized by cytogenetic analysis, FISH, or by Southern blot. However, in most cases the underlying mutations are too subtle to be revealed by these techniques and are best characterized by examination of PCR products generated from putative disease alleles. While there are over a dozen different methods to screen for mutant alleles in PCR products, no technique is absolutely sensitive and each has specific advantages and disadvantages in its ability to detect unknown mutations.

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References

  1. Orita, M., Suzuki, Y., Sekiya, T., and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879.

    Article  PubMed  CAS  Google Scholar 

  2. White, M. B., Carvalho, M., Derse, D., O’Brien, S. J., and Dean, M. (1992) Detecting single base substitutions as heteroduplex polymorphisms. Genomics 12, 301–306.

    Article  PubMed  CAS  Google Scholar 

  3. Cotton, R. G. H., Rodrigues, N. R., and Campbell, R. D. (1988) Reactivity of cytosine and thymine in single-base-pair mismatches with hydroxylamide and osmium tetroxide and its application to the study of mutations. Proc. Natl. Acad. Sci. USA 85, 4397–4401.

    Article  PubMed  CAS  Google Scholar 

  4. Myers, R. M., Maniatis, T., and Lerman, L. S. (1987) Detection and localization of single base changes by denaturing gradient gel electrophoresis. Meth. Enzymol. 155, 501–527.

    Article  PubMed  CAS  Google Scholar 

  5. Sheffield, V. C., Cox, D. R., Lerman, L. S., and Myers, R. M. (1989) Attachment of a 40-base-pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc. Natl. Acad. Sci. USA 86, 232–236.

    Article  PubMed  CAS  Google Scholar 

  6. Forrest, S. M., Dahl, H. H., Howells, D. W., Dianzani, I., and Cotton, R. G. H. (1991) Mutation detection in phenylketonuria by using chemical cleavage of mismatch: importance of using probes from both normal and patient samples. Am. J. Hum. Genet. 49, 175–183.

    PubMed  CAS  Google Scholar 

  7. Youil, R., Kemper, B. W., and Cotton, R. G. H. (1995) Screening for mutations by enzyme mismatch cleavage with T4 endonuclease VII. Proc. Natl. Acad. Sci. USA 92, 87–91.

    Article  PubMed  CAS  Google Scholar 

  8. Mashal, R. D., Koontz, J., and Sklar, J. (1995) Detection of mutations by cleavage of DNA heteroduplexes with bacteriophage resolvases. Nat. Genet. 9, 177–183.

    Article  PubMed  CAS  Google Scholar 

  9. Roest, P. A. M., Roberts, R. G., Sugino, S., Ommen, G. J. B., and Dunnen, J. T. (1993) Protein truncation test (PTT) for rapid detection of translation-terminating mutations. Hum. Mol. Genet. 2, 1719–1721.

    Article  PubMed  CAS  Google Scholar 

  10. Powell, S. M., Petersen., G. M., Krush, A. J., Booker, S., Jen, J., Giardiello, F. M., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. (1993) Molecular diagnosis of familial adenomatous polyposis. N. Engl. J. Med. 329, 1982–1987.

    Article  PubMed  CAS  Google Scholar 

  11. Sarkar, G. and Sommer, S. S. (1989) Access to a messenger RNA sequence or its protein product is not limited by tissue or species specificity. Science 244, 331–334.

    Article  PubMed  CAS  Google Scholar 

  12. Heim, R. A., Silverman, L. M., Farber, R. A., Kam-Morgan, L. N. W., and Luce, M. C. (1994) Screening for truncated NF1 proteins. Nature Genet. 8, 218,219.

    Article  PubMed  CAS  Google Scholar 

  13. Heim, R. A., Kam-Morgan, L. N. W., Binnie, C. G., Corns, D. D., Cayouette, M. C., Farber, R. A., Aylsworth, A. S., Silverman, L. M., and Luce, M. C. (1995) Distribution of 13 truncating mutations in the neurofibromatosis 1 gene. Hum. Mol. Genet. 4, 975–981.

    Article  PubMed  CAS  Google Scholar 

  14. Hogervorst, F. B. L., Cornelis, R. S., Bout, M., van Vliet, M., Oosterwijk, J. C., Olmer, R., Bakker, B., Klijn, J. G. M., Vasen, H. F. A., Meijers-Heijboer, H., Menko, F. H., Cornelisse, C. J., den Dunnen, J. T., Devilee, P., and van Ommen, G. J. B. (1995) Rapid detection of BRCA1 mutations by the protein truncation test. Nat. Genet. 10, 208–212.

    Article  PubMed  CAS  Google Scholar 

  15. Struewing, J. P., Brody, L. C., Erdos, M. R., Kase, R. G., Giambarresi, T. R., Smith, S. A., Collins, F. S., and Tucker, M. A. (1995) Detection of eight BRCA1 mutations in 10 breast/ovarian cancer families, including 1 family with male breast cancer. Am. J. Hum. Genet. 57, 1–7.

    PubMed  CAS  Google Scholar 

  16. Szabo, C. I. and King, M. C. (1995) Inherited breast and ovarian cancer. Hum. Mol. Genet. 4, 1811–1817.

    PubMed  CAS  Google Scholar 

  17. St. John, D. J. B., McDermott, F. T., Hopper, J. L., Debney, E. A., Johnson, W. R., and Hughes, E. S. R. (1993) Cancer risk in relatives of patients with common colorectal cancer. Ann. Intern. Med. 118, 785–790.

    PubMed  CAS  Google Scholar 

  18. Bailey-Wilson, J. E., Elston, R. C., Schuelke, G. S., Kimberling, W., Albano, W., Lynch, J. F., and Lynch, H. T. (1986) Segregation analysis of hereditary nonpolyposis colorectal cancer. Genet. Epidemiol. 3, 27–38.

    Article  PubMed  CAS  Google Scholar 

  19. Nagase, H. and Nakamura, Y. (1993) Mutations of the APC (adenomatous polyposis coli) gene. Hum. Mut. 2, 425–434.

    Article  PubMed  CAS  Google Scholar 

  20. Vasen, H. F. A., Mecklin, J. P., Meera Khan, P., and Lynch, H. T. (1991) The international collaborative group on hereditary non-polyposis colorectal cancer (ICG-HNPCC). Dis. Colon Rect. 34, 424–425.

    Google Scholar 

  21. Lynch, H. T., Smyrk, T. C., Watson, P., Lanspa, S. J., Lynch, J. F., Lynch, P. M., Cavalieri, R. J., and Boland, C. R. (1993) Genetics, natural history, tumor spectrum and pathology of hereditary nonpolyposis colorectal cancer: an updated review. Gastroenerology 104, 1535–1549.

    CAS  Google Scholar 

  22. Leach, F. S., Nicolaides, N. C., Papadopoulos, N., Liu, B., Jen, J., Parsons, R., Peltomaki, P., Sistonen, P., Aaltonen, L. A., Nystrom-Lahti, M., Guan, X. Y., Zhang, J., Meltzer, P. S., Yu, J. W., Kao, F. T., Chen, D. J., Cerosaletti, K. M., Fournier, R. E. K., Todd, S., Lewis, T., Leach, R. J., Naylor, S. L., Weissenbach, J., Mecklin, J. P., Jarvinen, H., Petersen, G. M., Hamilton, S. R., Green, J., Jass, J., Watson, P., Lynch, H. T., Trent, J. M., de la Chapelle, A., Kinzler, K. W., and Vogelstein, B. (1993) Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell 75, 1215–1225.

    Article  PubMed  CAS  Google Scholar 

  23. Bronner, C. E., Baker, S. M., Morrison, P. T., Warren, G., Smith, L. G., Lescoe, M. K., Kane, M., Earabino, C., Lipford, J., Lindblom, A., Tannergard, P., Bollag, R. J., Godwin, A. R., Ward, D. C., Nordenskjold, M., Fishel, R., Kolodner, R., and Liskay, R. M. (1994) Mutation in the DNA mismatch repair gene homologue hMLH1 is associated with hereditary non-polyposis colon cancer. Nature 368, 258–261.

    Article  PubMed  CAS  Google Scholar 

  24. Papadopoulos, N., Nicolaides, N. C., Wei, Y. F., Rubin, S. M., Carter, K. C., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, J. C., Hamilton, S. R., Petersen, G. M., Watson, P., Lynch, H. T., Peltomaki, P., Mecklin, J. P., de la Chapelle, A., Kinzler, K. W., and Vogelstein, B. (1994) Mutation of a mutL homolog in hereditary colon cancer. Science 263, 1625–1629.

    Article  PubMed  CAS  Google Scholar 

  25. Nicolaides, N. C., Papadopoulos, N., Liu, B., Wei, Y. F., Carter, K. C., Ruben, S. M., Rosen, C. A., Haseltine, W. A., Fleischmann, R. D., Fraser, C. M., Adams, M. D., Venter, J. C., Dunlop, M. G., Hamilton, S. R., Petersen, G. M., de la Chapelle, A., Vogelstein, B., and Kinzler, K. W. (1994) Mutations of two PMS homologues in hereditary nonpolyposis colon cancer. Nature 371, 75–80.

    Article  PubMed  CAS  Google Scholar 

  26. Hemminki, A., Peltomaki, P., Mecklin, J. P., Jarvinen, H., Salovaara, R., Nystrom-Lahti, M., de la Chapelle, A., and Aaltonen, L. A. (1994) Loss of the wild type MLH1 gene is a feature of hereditary nonpolyposis colorectal cancer. Nat. Genet. 8, 405–410.

    Article  PubMed  CAS  Google Scholar 

  27. Markowitz, S., Wang, J., Myeroff, L., Parsons, R., Sun, L., Lutterbaugh, J., Fan, R. S., Zborowska, E., Kinzler, K. W., Vogelstein, B., Brattain, M., and Willson, J. K. V. (1995) Inactivation of type II TGF-b receptor in colon cancer cells with microsatellite instability. Science 268, 1336–1338.

    Article  PubMed  CAS  Google Scholar 

  28. Luce, M. C., Marra, G., Chauhan, D. P., Laghi, L., Carethers, J. M., Cherian, S. P., Hawn, M., Binnie, C. G., Kam-Morgan, L. N. W., Cayouette, M. C., Koi, M., and Boland, C. R. (1995) In vitro transcription/translation assay for the screening of hMLH1 and hMSH2 mutations in familial colon cancer. Gastroenterology 109, 1368–1374.

    Article  PubMed  CAS  Google Scholar 

  29. Kozak, M. (1987) An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125–8133.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Myriad Genetics, Salt Lake City, UT

    Michael C. Luce & Cameron G. Binnie

  2. Department of Molecular Genetics, Laboratory Corporation of America, Research Triangle Park, NC

    Lauren N. W. Kam-Morgan & Matthew C. Cayouette

Authors
  1. Michael C. Luce
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  2. Cameron G. Binnie
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  3. Lauren N. W. Kam-Morgan
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  4. Matthew C. Cayouette
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Editor information

Editors and Affiliations

  1. University of Maryland School of Medicine, Baltimore, MD

    Stephen J. Meltzer MD

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© 1998 Humana Press Inc.

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Luce, M.C., Binnie, C.G., Kam-Morgan, L.N.W., Cayouette, M.C. (1998). In Vitro Transcription/Translation Analysis for the Identification of Translation-Terminating Mutations. In: Meltzer, S.J. (eds) PCR in Bioanalysis. Methods In Molecular Medicine™, vol 92. Humana Press. https://doi.org/10.1385/0-89603-497-6:127

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  • DOI: https://doi.org/10.1385/0-89603-497-6:127

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-497-6

  • Online ISBN: 978-1-59259-575-4

  • eBook Packages: Springer Protocols

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