Repeat Analysis Pooled Isolation and Detection (RAPID) Cloning of Microsatellite Expansions

  • Laura P. W. Ranum
Part of the Methods in Molecular Biology™ book series (MIMB, volume 217)


Microsatellite repeat expansions have been shown to cause a number of neurodegenerative diseases (1). Most of the disease genes identified to date involve the expansion of a trinucleotide repeat motif, but recently tetra- and pentanucleotide repeat expansions have been shown to cause myotonic dystrophy type 2 (DM2) and spinocerebellar ataxiatype 10 (SCA10), respectively (2,3). Most microsatellite diseases are characterized by the presence of anticipation, or a decrease in the age of onset in consecutive generations due to the tendency of the unstable repeat tract to lengthen when passed from one generation to the next (1,4,5). In addition, the involvement of trinucleotide repeat expansions in a number of other diseases including schizophrenia (6) and bipolar affective disorder (7,8) has been suggested both by the presence of anticipation and by Repeat Expansion Detection (RED) analysis (9,10). The involvement of trinucleotide expansions in these diseases, however, can only be conclusively confirmed by the isolation of the expansions present in these populations and detailed analysis to assess each expansion as a possible pathogenic mutation. We previously described a novel procedure to quickly isolate expanded trinucleotide repeats and the corresponding flanking nucleotide sequence directly from small amounts of genomic DNA using a process of Repeat Analysis, Pooled Isolation, and Detection of individual clones containing expanded trinucleotide repeats (RAPID cloning) (11). We used this technology to clone the pathogenic SCA7 and SCA8 CAG/CTG repeat expansions from banked DNA samples from single individuals affected with ataxia (11, 12, 13).


Repeat Expansion Trinucleotide Repeat Bipolar Affective Disorder Lambda Phage Helper Phage 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Brice, A. (1998) Unstable mutations and neurodegenerative disorders. J. Neurol. 245(8), 505–510.PubMedCrossRefGoogle Scholar
  2. 2.
    Liquori, C., Ricker, K., Moseley, M. L., Jacobsen, J. F., Kress, W., Naylor, S., Day, J. W., and Ranum, L. P. W. (2001) Myotonic dystrophy type 2 caused by a CCTG expansion in intron 1 of ZNF9. Science 293, 864–867.PubMedCrossRefGoogle Scholar
  3. 3.
    Matsuura, T., Yamagata, T., Burgess, D. L., Rasmussen, A., Grewal, R. P., Watase, K. (2000) Large expansion of the ATTCT pentanucleotide repeat in spinocerebellar ataxia type 10. Nat. Genet. 26(2), 191–194.PubMedCrossRefGoogle Scholar
  4. 4.
    Warren, S.T. (1996) The expanding world of trinucleotide repeats. Science 271, 1374–1375.PubMedCrossRefGoogle Scholar
  5. 5.
    Klockgether, T. and Evert, B. (1998) Genes involved in hereditary ataxias. Trends Neurosci. 21(9), 413–418.PubMedCrossRefGoogle Scholar
  6. 6.
    O’Donovan, M., Guy, C., Craddock, N., Murphy, K., Cardno, A., Jones, L., et al. (1995) Expanded CAG repeats in schizophrenia and bipolar disorder. Nat. Genet. 10, 380–381.CrossRefGoogle Scholar
  7. 7.
    Oruc, L., Lindblad, K., Verheyen, G., Ahlberg, S., Jakovljevic, M., Ivezic, S., et al. (1997) CAG repeat expansions in bipolar and unipolar disorders. Am. J. Hum. Genet. 60, 732–735.Google Scholar
  8. 8.
    Vincent, J. B., Paterson, A. D., Strong, E., Petronis, A., and Kennedy, J. L. (2000) The unstable trinucleotide repeat story of major psychosis. Am. J. Med. Genet. 97(1), 77–97.PubMedCrossRefGoogle Scholar
  9. 9.
    Schalling, M., Hudson, T., Buetow, K., and Housman, D. (1993) Direct detection of novel expanded trinucleotide repeats in the human genome. Nat. Genet. 4, 135–139.PubMedCrossRefGoogle Scholar
  10. 10.
    Lindblad, K., Zander, C., Schalling, M., and Hudson, T. (1994) Growing triplet repeats. Nat. Genet. 7, 124.PubMedCrossRefGoogle Scholar
  11. 11.
    Koob, M. D., Benzow, K. A., Bird, T. D., Day, J. W., Moseley, M. L., and Ranum, L. P. W. (1998) Rapid cloning of expanded trinucleotide repeat sequences from genomic DNA. Nat. Genet. 18, 72–75.PubMedCrossRefGoogle Scholar
  12. 12.
    Moseley, M. L., Benzow, K. A., Schut, L. J., Bird, T. D., Gomez, C. M., Barkhaus, P. E. (1998) Incidence of dominant spinocerebellar and Friedreich triplet repeats among 361 ataxia families. Neurology 51(6), 1666–1671.Google Scholar
  13. 13.
    Koob, M.D., Moseley, M. L., Schut, L. J., Benzow, K. A., Bird, T. D., Day, J. W., and Ranum, L. P. W. (1999) An untranslated CTG expansion causes a novel form of spinocer-ebellar ataxia (SCA8). Nat. Genet. 21(4), 379–384.Google Scholar
  14. 14.
    Holmes, S. E., O’Hearn, E. E., McInnis, M. G., Gorelick-Feldman, D. A., Kleiderlein, J. J., Callahan, C. (1999) Expansion of a novel CAG trinucleotide repeat in the 5′ region of PPP2R2B is associated with SCA12. Nat. Genet. 23(4), 391–392.PubMedCrossRefGoogle Scholar
  15. 15.
    Holmes, S. E., O’Hearn, E. E., Rosenblatt, A., Callahan, C., Hwang, H., Ingersoll-Ashworth, R. G., et al. (2001) A repeat expansion in the gene encoding juctophillin-3 is associated with Huntington disease-like 2. Nat. Genet. 29, 377–378.PubMedCrossRefGoogle Scholar
  16. 16.
    Ostrander, E. O., Jong, P. M., Rine J., and Duyk, G. (1992) Construction of small-insert genomic DNA libraries highly enriched for microsatellite repeat sequences. Proc. Natl. Acad. Sci. USA 89, 3419–3423.PubMedCrossRefGoogle Scholar
  17. 17.
    Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 154, 367–382.PubMedCrossRefGoogle Scholar
  18. 18.
    Nakamoto, M., Takebayashi, H., Kawaguchi, Y., Narumiya, S., Taniwaki, M., Nakamura, Y., et al. (1997) A CAG/CTG expansion in the normal population. Nat. Genet. 17(4), 385–386.Google Scholar
  19. 19.
    Breschel, T. S., McInnis, M. G., Margolis, R. L., Sirugo, G., Corneliussen, B., Simpson, S. G., et al. (1997) A novel, heritable, expanding CTG repeat in an intron of the SEF2-1 gene on chromosome 18q21.1. Hum. Mol. Genet. 6(11), 1855–1863.Google Scholar

Copyright information

© Humana Press Inc. 2003

Authors and Affiliations

  • Laura P. W. Ranum
    • 1
  1. 1.Department of Genetics, Cell Biology, and Development, Institute of Human GeneticsUniversity of MinnesotaMinneapolis

Personalised recommendations