Advertisement

FISH With and Without COT1 DNA

  • Vladimir A. TrifonovEmail author
  • Nadezhda N. Vorobieva
  • Willem Rens
Protocol

Complex FISH probes comprising large spans of genomic DNA always contain a high amount of dispersed repetitive sequences that hamper the visualization of the specific signal of interest. To overcome this problem, different approaches have been elaborated that depend on the type of experiment and the quality of the probe. A classical way to suppress repetitive sequences is to use unlabeled competitor DNA (sheared total genomic DNA or repeated sequence enriched DNA fractions). Here we present two protocols: the first one describes rapid COT DNA isolation and the peculiarities of its use in different FISH experiments, and the second is used for COT-free FISH with complex probes and is based on a special software tool for image enhancement.

Keywords

Repetitive Sequence Chromosome Paint Yellow Pixel Disperse Repetitive Sequence Image Enhancement Tool 
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.

Notes

Acknowledgments

This work was supported by a grant of the RFBR, DFG research grants to VAT and NVV, and by a Welcome Trust grant to WR.

References

  1. Britten RJ, Graham DE, Neufeld BR (1974) Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 29:363–419CrossRefPubMedGoogle Scholar
  2. Bolzer A, Craig JM, Cremer T, Speicher MR (1999) A complete set of repeat-depleted, PCR- amplifiable, human chromosome-specific painting probes. Cytogenet Cell Genet 84:233–240CrossRefPubMedGoogle Scholar
  3. Craig JM, Kraus J, Cremer T (1997) Removal of repetitive sequences from FISH probes using PCR-assisted affinity chromatography. Hum Genet 100:472–476CrossRefPubMedGoogle Scholar
  4. Dugan LC, Pattee MS, Williams J, Eklund M, Sorensen K, Bedford JS, Christian AT (2005) Polymerase chain reaction-based suppression of repetitive sequences in whole chromosome painting probes for FISH. Chromosome Res 13:27–32CrossRefPubMedGoogle Scholar
  5. Hopman AHM., Raap AK, Landegent JE, Wiegant J, Boerman RH (1988) Nonradioactive in situ hybridization. Molec Neuroanat 10:43–64Google Scholar
  6. Landegent JE, Jansen in de Wal N, Dirks RW, van der Ploeg M (1987) Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Human Genet 77:366–370CrossRefGoogle Scholar
  7. Lichter P, Cremer T, Borden J, Manuelidis L, Ward DC (1988) Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet 80:224–234CrossRefPubMedGoogle Scholar
  8. Pardue ML, Gall JG (1975) Nucleic acid hybridization to the DNA of cytological preparations. Methods Cell Biol 10:1–16CrossRefPubMedGoogle Scholar
  9. Rabbitts P, Impey H, Heppell-Parton A, Langford C, Tease C, Lowe N, Bailey D, Malcolm Ferguson-Smith MA, Carter N (1995) Chromosome specific paints from a high resolution flow karyotype of the mouse. Nat Genet 9:369–375CrossRefPubMedGoogle Scholar
  10. Rens W, Grützner F, O'Brien PC, Fairclough H, Graves JA, Ferguson-Smith MA (2004) Resolution and evolution of the duck-billed platypus karyotype with an X1Y1X2Y2X3Y3X4- Y4X5Y5 male sex chromosome constitution. Proc Natl Acad Sci USA 101:16257–16261CrossRefPubMedGoogle Scholar
  11. Rens W, Moderegger K, Skelton H, Clarke O, Trifonov V, Ferguson-Smith MA (2006) A procedure for image enhancement in chromosome painting. Chromosome Res 14:497–503CrossRefPubMedGoogle Scholar
  12. Rogan PK, Cazcarro PM, Knoll JH (2001) Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization. Genome Res 11:1086–1094CrossRefPubMedGoogle Scholar
  13. Sealey PG, Whittaker PA, Southern EM (1985) Removal of repeated sequences from hybridisation probes. Nucleic Acids Res 13:1905–1922CrossRefPubMedGoogle Scholar
  14. Telenius HC, Carter NP, Bebb CE et al. (1992) Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer. Genomics 13:718–725CrossRefPubMedGoogle Scholar
  15. Wienberg J, Adamski E, Yang F, Müller S, Ferguson-Smith MA (1997) Chromosome painting without competitor DNA. Trends Genet Tech Tips Online (homepage at http://www1.elsevier.com/homepage/sab/tto/menu.htm)
  16. Yang F, O'Brien P, Milne B, Graphodatsky A, Solansky N, Trifonov V, Rens W, Sargan D, Ferguson-Smith MA (1999) A complete comparative chromosome map for the dog, red fox, and and human and its integration with canine genetic maps. Genomics 62:189–202CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2009

Authors and Affiliations

  • Vladimir A. Trifonov
    • 1
    Email author
  • Nadezhda N. Vorobieva
    • 1
  • Willem Rens
    • 2
  1. 1.Institute of Cytology and GeneticsRussian Academy of SciencesNovosibirskRussian Federation
  2. 2.Department of Veterinary MedicineUniversity of CambridgeCambridgeUK

Personalised recommendations