Skip to main content

Fluorescent In Situ Hybridization of DNA Probes in the Interphase and Metaphase Stages of the Cell Cycle

  • Protocol
  • First Online:
Basic Cell Culture Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 946))

Abstract

In the past decade, fluorescent in situ hybridization (FISH) has been used routinely in detecting molecular abnormalities in the interphase and metaphase stages of the cell cycle. Many of the molecular anomalies which are detected in this manner are diagnostic of a prenatal, postnatal, or neoplastic genetic disorder. With the continuous isolation of commercially available DNA probes specific to a particular chromosome region, FISH analysis has become standardized in its ability to detect characteristic chromosomal anomalies in association with genetic and neoplastic diseases. In recent years, FISH has also become automated to accommodate the increased volume of slide preparations necessary for the number of DNA probes needed to detect characteristic molecular anomalies in cancer tissues and bone marrow samples. FISH technology provides essential information to the physician regarding the diagnosis, response to treatment, and ultimately the prognosis of their patients’ disorder. It has become an important source of information routinely used in conjunction with chromosome analyses, and presently to confirm molecular alterations detected by array comparative genomic hybridization (aCGH) analyses. In this chapter we describe the methods for performing FISH analyses in order to determine the presence or the absence of genetic abnormalities which define whether the patient has either a genetic syndrome or malignant disease.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 139.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 179.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Lichter P, Cremer T, Tang C-JC, Watkins PC, Manuelidis L (1988) Rapid detection of human chromosome 21 aberrations by in situ hybridization. Proc Natl Acad Sci USA 85:9664–9668

    Article  PubMed  CAS  Google Scholar 

  2. Singer RH, Lawrence JB, Villnave C (1986) Optimization of in situ hybridization using isotopic and non-isotopic detection methods. Biotechniques 4:230

    CAS  Google Scholar 

  3. Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83:2934–2938

    Article  PubMed  CAS  Google Scholar 

  4. Tkachuk DC, Westbrook CA, Andreeff M, Donlon TA, Cleary ML, Suryanarayan K, Homge M, Redner A, Gray J, Pinkel D (1990) Detection of bcr-abl fusion in chronic myelogeneous leukemia by in situ hybridization. Science 250:559–562

    Article  PubMed  CAS  Google Scholar 

  5. Cannizzaro LA (1997) Special techniques in cytogenetics. In: Wolman SR, Sell S (eds) Cytogenetic markers of human cancer. Humana, NY pp 461–477

    Google Scholar 

  6. Cannizzaro LA, Shi G (1997) Fluorescent in situ hybridization. In: Walker J, Pollard J (eds) Animal cell culture. Methods in molecular biology, vol 25. Humana, NY pp 313–322

    Google Scholar 

  7. Poddighe EJ, Moesker O, Smeets D, Awwad BH, Ramackers FCS, Hopman AHN (1993) Interphase cytogenetics of hematological cancer: comparison of classical karyotyping and in situ hybridization using a panel of eleven chromosome specific DNA probes. Cancer Res 51:1959–1967

    Google Scholar 

  8. Xing Y, Johnson CV, Dobner ER, Lawrence JB (1993) Higher level organization of individual gene transcription and RNA splicing. Science 259:1326–1335

    Article  PubMed  CAS  Google Scholar 

  9. Rupa DS, Hasegawa L, Eastmond DA (1995) Detection of chromosomal breakage in the lcen-lql2 region of interphase human lymphocytes using multicolor fluorescence in situ hybridization with tandem DNA probes. Cancer Res 55:640–645

    PubMed  CAS  Google Scholar 

  10. Ariyama T, Inazawa J, Ezaki T, Nakamura Y, Horii A, Abe T (1995) High-resolution cytogenetic mapping of the short arm of chromosome 1 with newly isolated 41 l cosmid markers by fluorescence in situ hybridization: the precise order of 18 markers on lp36.1 on prophase chromosomes and “stretched” DNAs. Genomics 25:114–123

    Article  PubMed  CAS  Google Scholar 

  11. Trask B, Pinkel D, Van Den Engh G (1989) The proximity of DNA sequences in interphase cell nuclei is correlated to genomic distance and permits ordering ofcosmids spanning 250 kilobase pairs. Genomics 5:710–717

    Article  PubMed  CAS  Google Scholar 

  12. Lawrence JB, Singer RH, McNeil JA (1990) Interphase and metaphase resolution of different distances within the human dystrophin gene. Science 249:928–932

    Article  PubMed  CAS  Google Scholar 

  13. Brandriff B, Gordon L, Trask B (1991) A new system for high-resolution DNA sequence mapping in interphase pronuclei. Genomics 10:75–82

    Article  PubMed  CAS  Google Scholar 

  14. Lawrence JB, Singer RH (1991) Spatial organization of nucleic acid sequences within cells. Sem Cell Biol 2:82–101

    Google Scholar 

  15. Cannizzaro LA (1991) Gene mapping in cancer. Cancer Genet Cytogenet 55:139–147

    Article  PubMed  CAS  Google Scholar 

  16. Shi G, Cannizzaro LA (1996) Mapping of 29 YAC clones and identification of 3 YACs spanning the translocation t(3;8)(p14.2;q24.1) breakpoint at 8q24.1 in hereditary renal cell carcinoma. Cytogenet Cell Genet 75:180–185

    Article  PubMed  CAS  Google Scholar 

  17. Engel H, Drach J, Keyhani A, Jiang S, Van NT, Kimmel M, Sanchez-Williams G, Coodacre A, Andreef M (1999) Quantitation of minimal residual disease in acute myelogenous leukemia and myelodysplastic syndromes in complete remission by molecular cytogenetics of progrenitor cells. Leukemia 13(4):568–577

    Article  PubMed  CAS  Google Scholar 

  18. Tanaka K, Arif M, Eguchi M, Shintani T, Kumaravel TS, Asaoku H, Kyo T, Dohy H, Kamada N (1999) Interphase fluorescence in situ hybridization overcomes pitfalls of G-banding analysis with special reference to underestimation of chromosome aberration rates. Cancer Genet Cytogenet 115(1):32–38

    Article  PubMed  CAS  Google Scholar 

  19. Rigolin GM, Bigoni R, Milani R, Cavazzini F, Roberti MG, Bardi A, Agostini P, Della Porta M, Teighi A, Piva N, Cuneo A, Castoldi G (2001) Clinical importance of interphase cytogenetics detecting occult chromosome lesions in myelodysplastic syndromes with normal karyotype. Leukemia 15(12):1841–1847

    Article  PubMed  CAS  Google Scholar 

  20. Ishikawa M, Yagasaki F, Okamura D, Maeda T, Sugahara Y, Jinnai I, Bessho M (2007) A novel gene, ANKRD28 on 3p25, is fused with NUP98 on 11p15 in a cryptic 3-way translocation of t(3;5;11)(p25;q35;p15) in an adult patient with myelodysplastic syndrome/acute myelogenous leukemia. Int J Hematol 86(3):238–245

    Article  PubMed  CAS  Google Scholar 

  21. Barouk-Simonet E, Soenen-Cornu V, Roumier C, Cosson A, Lai JL, Fenaux P, Preudhomme C (2005) Role of multiplex FISH in identifying chromosome involvement in myelodysplastic syndromes and acute myeloid leukemias with complex karyotypes: a report of 28 cases. Cancer Genet Cytogenet 157(2):118–126

    Article  PubMed  CAS  Google Scholar 

  22. Papenhausen PR, Griffin S, Tepperberg J (2005) Oncogene amplification in transforming myelodysplasia. Exp Mol Pathol 79(2):168–175

    Article  PubMed  CAS  Google Scholar 

  23. Phan CL, Megat Baharuddin PJ, Chin LP, Zakaria Z, Yegappan S, Sathar J, Tan SM, Purushothaman V, Chang KM (2008) Amplification of BCR-ABL and t(3;21) in a patient with blast crisis of chronic myelogenous leukemia. Cancer Genet Cytogenet 180(1):60–64

    Article  PubMed  CAS  Google Scholar 

  24. Lugthart S, van Drunen E, van Norden Y, van Hoven A, Erpelinch CA, Valk PJ, Beverloo HB, Lowenberg B, Delwel R (2008) High EVI1 levels predict adverse outcome in acute myeloid leukemia: prevalence of EVI1 overexpression and chromosome 3q26 abnormalities underestimated. Blood 111(8):4329–4337

    Article  PubMed  CAS  Google Scholar 

  25. Mrozek K (2008) Cytogenetic, molecular genetic, and clinical characteristics of acute myeloid leukemia with a complex karyotype. Simin Oncol 35(4):365–377

    Article  CAS  Google Scholar 

  26. Schoch C, Haferlach T, Bursch S, Gerstner D, Schnittger S, Dugas M, Kern W, Loffler H, Hiddemann W (2002) Loss of genetic material is more common than gain in acute myeloid leukemia with complex aberrant karyotype: a detailed analysis of 125 cases using conventional chromosome analysis and fluorescence in situ hybridization including 24-color FISH. Genes Chromosomes Cancer 35(1):20–29

    Article  PubMed  Google Scholar 

  27. Frohling S, Kayser S, Mayer C, Miller S, Wieland C, Skelin S, Schlenk RF, Dohner H, Dohner K (2005) Diagnostic value of fluorescence in situ hybridization for the detection of genomic aberrations in older patients with acute myeloid leukemia. Haematologica 90(2):194–199

    PubMed  Google Scholar 

  28. Ohsaka A, Otsubo K, Yokota H, Hisa T, Saito H, Kozaki T (2008) Spectral karyotyping and fluorescence in situ hybridization analyses identified a novel three-way translocation involving inversion 16 in therapy-related acute myeloid leukemia M4eo. Cancer Genet Cytogenet 184(2):113–118

    Article  PubMed  CAS  Google Scholar 

  29. Seller MJ, Bint S, Kavalier F, Brown RN, Ogilvie CM (2006) Multicolor banding detects a complex three chromosome, seven breakpoint unbalanced rearrangement in an ICSI-derived fetus with multiple abnormalities. Am J Med Genet A 140(10):1102–1107

    PubMed  Google Scholar 

  30. Moosavi SA, Sanchez J, Adeyinka A (2009) Marker chromosomes are a significant mechanism of high-level RUNX1 gene amplification in hematologic malignancies. Cancer Genet Cytogenet 189(1):24–28

    Article  PubMed  CAS  Google Scholar 

  31. Maitta RW, Cannizzaro LA, Ramesh KH (2009) Association of MLL amplification with poor outcome in acute myeloid leukemia. Cancer Genet Cytogenet 192:40–43

    Article  PubMed  CAS  Google Scholar 

  32. Cox MC, Panetta P, Venditti A, Del Poeta G, Franchi A, Buccisano F, Tamburini A, Maurillo L, Amadori S (2003) Comparison between conventional banding analysis and FISH screening with an AML-specific set of probes in 260 patients. Hematol J 4(4):263–270

    Article  PubMed  CAS  Google Scholar 

  33. Italiano A, Attias R, Aurias A, Perot G, Burel-Vandenbos F, Otto J, Venissac N, Pedeutour F (2006) Molecular cytogenetic characterization of a metastatic lung sarcomatoid carcinoma: 9p23 neocentromere and 9p23-p24 amplification including JAK2 and JMJD2C. Cancer Genet Cytogenet 167(2):122–130

    Article  PubMed  CAS  Google Scholar 

  34. Fenaux P (2001) Chromosome and molecular abnormalities in myelodysplastic syndromes. Int J Hematol 73(4):429–437

    Article  PubMed  CAS  Google Scholar 

  35. Cheng L, Ramesh KH, Wei D, Ratech H, Radel E, Cannizzaro LA (2001) t(11;19)(q23;p13.3) rearrangement in a patient with therapy related acute myeloid leukemia. Cancer Genet Cytogenet 129:17–22

    Article  PubMed  CAS  Google Scholar 

  36. Castuma MV, Rao PH, Acevedo SH, Larripa IB (2000) Comparative genomic hybridization study of de novo myeloid neoplasia. Acta Haematol 104(1):25–30

    Article  PubMed  CAS  Google Scholar 

  37. Baloglu H, Cannizzaro LA, Jones J, Koss LG (2001) Atypyical endometrial hyperplasia shares genomic abnormalities with endometrioid carcinoma by comparative genomic hybridization. Hum Pathol 32:615–622

    Article  PubMed  CAS  Google Scholar 

  38. Suela J, Alvarez S, Cigudosa JC (2007) DNA profiling by arrayCGH in acute myeloid leukemia and myelodysplastic syndromes. Cytogenet Genome Res 118(2–4):304–309

    Article  PubMed  CAS  Google Scholar 

  39. Evers C, Beier M, Poelitz A, Hildebrandt B, Servan K, Drechsler M, Germing U, Royer HD, Royer-Pokora B (2007) Molecular definition of chromosome arm 5q deletion end points and detection of hidden aberrations in patients with myelodysplastic syndromes and isolated del(5q) using oligonucleotide array CGH. Genes Chromosomes Cancer 46(12):1119–1128

    Article  PubMed  CAS  Google Scholar 

  40. Shaffer LG, Tommerup N (eds) (2005) ISCN 2005: an international system for human cytogenetic nomenclature. S. Karger, Basel

    Google Scholar 

  41. American College of Medical Genetics, Standards and Guidelines for Clinical Genetics Laboratories. 2006 Edition. 9. E. http://www.acmg.net/

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Linda A. Cannizzaro .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2013 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Cannizzaro, L.A. (2013). Fluorescent In Situ Hybridization of DNA Probes in the Interphase and Metaphase Stages of the Cell Cycle. In: Helgason, C., Miller, C. (eds) Basic Cell Culture Protocols. Methods in Molecular Biology, vol 946. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-128-8_5

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-128-8_5

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-127-1

  • Online ISBN: 978-1-62703-128-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics