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FISH with and Without COT1 DNA

  • Vladimir A. TrifonovEmail author
  • Nadezhda V. Vorobieva
  • Natalia A. Serdyukova
  • Willem Rens
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

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

Keywords

Repetitive DNA DNA reassociation kinetics Genome composition COT1-DNA 

Notes

Acknowledgments

This work was supported by grant of RSF (№14-14-00275) to VAT and by Welcome Trust grant to WR.

References

  1. 1.
    Pardue ML, Gall JG (1975) Nucleic acid hybridization to the DNA of cytological preparations. Methods Cell Biol 10:1–16CrossRefPubMedGoogle Scholar
  2. 2.
    Hopman AHM, Raap AK, Landegent JE et al (1988) Nonradioactive in situ hybridization. Mol Neuroanat 10:43–64Google Scholar
  3. 3.
    Sealey PG, Whittaker PA, Southern EM (1985) Removal of repeated sequences from hybridisation probes. Nucleic Acids Res 13:1905–1922CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Landegent JE, Jansen in de Wal N, Dirks RW et al (1987) Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum Genet 77:366–370CrossRefPubMedGoogle Scholar
  5. 5.
    Lichter P, Cremer T, Borden J et al (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
  6. 6.
    Britten RJ, Graham DE, Neufeld BR (1974) Analysis of repeating DNA sequences by reassociation. In: Methods in Enzymology. Academic Press, New York, pp 363–406Google Scholar
  7. 7.
    Rogan PK, Cazcarro PM, Knoll JH (2001) Sequence-based design of single-copy genomic DNA probes for fluorescence in situ hybridization. Genome Res 11:1086–1094CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    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
  9. 9.
    Bolzer A, Craig JM, Cremer T et al (1999) A complete set of repeat-depleted, PCR-amplifiable, human chromosome-specific painting probes. Cytogenet Cell Genet 84:233–240CrossRefPubMedGoogle Scholar
  10. 10.
    Dugan LC, Pattee MS, Williams J et al (2005) Polymerase chain reaction-based suppression of repetitive sequences in whole chromosome painting probes for FISH. Chromosome Res 13:27–32CrossRefPubMedGoogle Scholar
  11. 11.
    Wienberg J, Adamski E, Yang F et al (1997) Chromosome painting without competitor DNA. Trends Genet Technical Tips Online. http://www.elsevier.nl/locate/tto
  12. 12.
    Telenius H, 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
  13. 13.
    Rabbitts P, Impey H, Heppell-Parton A et al (1995) Chromosome specific paints from a high resolution flow karyotype of the mouse. Nat Genet 9:369–375CrossRefPubMedGoogle Scholar
  14. 14.
    Romanenko SA, Perelman PL, Trifonov VA et al (2015) A first generation comparative chromosome map between guinea pig (Cavia porcellus) and humans. PLoS ONE 10:e0127937CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Rens W, Moderegger K, Skelton H et al (2006) A procedure for image enhancement in chromosome painting. Chromosome Res 14:497–503CrossRefPubMedGoogle Scholar
  16. 16.
    Rens W, Grützner F, O’Brien PC et al (2004) Resolution and evolution of the duck-billed platypus karyotype with an X1Y1X2Y2X3Y3X4-Y4X5Y5 male sex chromosome constitution. Proc Natl Acad Sci U S A 101:16257–16261CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Yang F, O’Brien P, Milne B et al (1999) A complete comparative chromosome map for the dog, red fox, and human and its integration with canine genetic maps. Genomics 62:189–202CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Vladimir A. Trifonov
    • 1
    Email author
  • Nadezhda V. Vorobieva
    • 1
  • Natalia A. Serdyukova
    • 1
  • Willem Rens
    • 2
  1. 1.Institute of Molecular and Cellular Biology SB RASNovosibirskRussian Federation
  2. 2.Department of Veterinary MedicineUniversity of CambridgeCambridgeUnited Kingdom

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