In Situ Hybridization

  • Orlando J. Miller
  • Eeva Therman


Afascinating property of DNA is the complementarity of the nucleotide bases in its two anti-parallel strands, with G always pairing with C and A always pairing with T. This does not involve strong covalent chemical bonds but weak hydrogen bonds. There are three hydrogen bonds between G-C pairs and two between A-T pairs, so strand separation is easier in AT-rich DNA than in GC-rich DNA. Mild heating breaks these hydrogen bonds and is one way to separate the two strands, called denaturation or dissociation. Reducing the temperature under the right salt conditions leads to renaturation (reassociation or reannealing) of the two strands by reconstitution of the hydrogen bonds. The rate of renaturation depends on the frequency of collision between complementary sequences, which depends on their concentration. The concentration and time required for renaturation determines the Cot value (concentration × time). If a high concentration of labeled probe DNA is used, hybridization to complementary nucleic acid sequences in the target preparation can be achieved in a reasonably short time. These properties of DNA are extremely important, because they make it possible to detect specific DNA sequences (such as genes) on a nitrocellulose filter (molecular hybridization) or in cytological preparations (in situ hybridization) by using labeled DNA or RNA probes.


Metaphase Spread Pericentric Inversion Painting Probe Cytogenet Cell Spectral Karyotyping 
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.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Antonacci R, Marzella R, Finelli P, et al. (1995) A panel of subchromosomal painting libraries representing over 300 regions of the human genome. Cytogenet Cell Genet 68:25–32PubMedCrossRefGoogle Scholar
  2. Bailey SM, Meyne J, Cornforth MN, et al. (1996) A new method for detecting pericentric inversions using COD-FISH. Cytogenet Cell Genet 75: 248–253PubMedCrossRefGoogle Scholar
  3. Boggs BA, Chinault AC (1994) Analysis of replication timing of human X-chromosomal loci by fluorescence in situ hybridization. Proc Natl Acad Sci USA 91:6083–6087PubMedCrossRefGoogle Scholar
  4. Burke DT, Carle GF, Olson MV (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236:806–812PubMedCrossRefGoogle Scholar
  5. Chen Z, Grebe TA, Guan X-Y, et al. (1997) Maternal balanced translocation leading to partial duplication of 4q and partial deletion of 1p in a son: cytogenetic and FISH studies using band-specific painting probes generated by chromosome microdissection. Am J Med Genet 71: 160–166PubMedCrossRefGoogle Scholar
  6. Cremer T, Lichter P, Borden J, et al. (1988) Detection of chromosome aberrations in metaphase and interphase tumour cells by in situ hybridization using chromosome specific library probes. Hum Genet 80:235–246PubMedCrossRefGoogle Scholar
  7. Cullen DF, Yip M-Y, Eyre HJ (1997) Rapid detection of euchromatin by Alu-PRINS: use in clinical cytogenetics. Chrom Res 5:81–85CrossRefGoogle Scholar
  8. DeCapoa A, Felli MP, Baldini A, et al. (1988) Relationship between the numbers and function of human ribosomal genes. Hum Genet 79:301–304Google Scholar
  9. Femino AM, Fay FS, Fogarty K, et al. (1998) Visualization of single RNA transcripts in situ. Science 280:585–590PubMedCrossRefGoogle Scholar
  10. Forozan F, Karhu R, Kononen J, et al. (1997) Genome screening by comparative genomic hybridization. Trends Genet 13:405–409PubMedCrossRefGoogle Scholar
  11. Gartler SM, Goldstein L, Tyler-Freer SE, et al. (1999) The timing of XIST replication: dominance of the domain. Hum Mol Genet 8:1085–1089PubMedCrossRefGoogle Scholar
  12. Gläser B, Yen PH, Schempp W (1998) Fibre-FISH unravels apparently seven DAZ genes or pseudogenes clustered within a Y-chromosome region frequently deleted in azoospermic males. Chrom Res 6:481–486PubMedCrossRefGoogle Scholar
  13. Gosden J, Lawson D (1994) Rapid chromosome identification by oligonucleotide-primed in situ DNA synthesis (PRINS). Hum Mol Genet 3: 931–936PubMedCrossRefGoogle Scholar
  14. Harper ME, Saunders GF (1984) Localization of single-copy genes on human chromosomes by in situ hybridization of 3H-probes and autoradiography. In: Sparkes RS, de la Cruz FF (eds) Research perspectives in cytogenetics. University Park Press, Baltimore, pp 117–133Google Scholar
  15. Knight SJL, Horsley SW, Regan R, et al. (1997) Development and clinical application of an innovative fluorescence in situ hybridization technique which detects submicroscopic rearrangements involving telomeres. Eur J Hum Genet 5:1–8PubMedGoogle Scholar
  16. Kuwano A, Mutirangura A, Dittrich B, et al. (1992) Molecular dissection of the Prader-Willi/Angelman syndrome region (15q 11–13) by YAC cloning and FISH analysis. Hum Mol Genet 1:417–425PubMedCrossRefGoogle Scholar
  17. Levy B, Gershin IF, Desnick RJ, et al. (1997) Characterization of a de novo unbalanced chromosome rearrangement by comparative genomic hybridization and fluorescence in situ hybridization. Cytogenet Cell Genet 76:68–71PubMedCrossRefGoogle Scholar
  18. Lichter P, Ward DC (1990) Is non-isotopic in-situ hybridization finally coming of age? Nature 345:93–94PubMedCrossRefGoogle Scholar
  19. Lüdecke H-J, Senger G, Claussen U, et al. (1989) Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature 338:348–350PubMedCrossRefGoogle Scholar
  20. Mann SM, Burkin DJ, Grin DK, et al. (1997) A fast, novel approach for DNA fibre-fluorescence in situ hybridization. Chrom Res 5:145–147PubMedCrossRefGoogle Scholar
  21. Müller S, O’Brien PCM, Ferguson-Smith MA, et al. (1997a) A novel source of highly specific chromosome painting probes for human karyotype analysis derived from primate homologues. Hum Genet 101:149–153PubMedCrossRefGoogle Scholar
  22. Müller S, Rocchi M, Ferguson-Smith MA, et al. (1997b) Toward a multicolor chromosome bar code for the entire human karyotype by fluorescence in situ hybridization. Hum Genet 100:271–278PubMedCrossRefGoogle Scholar
  23. Pinkel D, Landegent J, Collins C, et al. (1988) Fluorescent in situ hybridization with human chromosome specific libraries: detection of trisomy 21 and translocation of chromosome 4. Proc Natl Acad Sci USA 85:9138–9142PubMedCrossRefGoogle Scholar
  24. Popp S, Jauch A, Schindler D, et al. (1993) A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones. Hum Genet 92:527–532PubMedCrossRefGoogle Scholar
  25. Schröck E, du Manoir S, Veldman T, et al. (1996) Multicolor spectral karyotyping of human chromosomes. Science 273:494–497PubMedCrossRefGoogle Scholar
  26. Selig S, Okumura K, Ward DC, et al. (1992) Delineation of DNA replication time zones by fluorescence in situ hybridization. EMBO J 11:1217–1225PubMedGoogle Scholar
  27. Shiels C, Coutelle C, Huxley C (1997) Analysis of ribosomal and alphoid repetitive DNA by fiber-FISH. Cytogenet Cell Genet 76:20–22PubMedCrossRefGoogle Scholar
  28. Shizuya H, Birren B, Kim U-J, et al. (1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherischia coli using an F-factor-based vector. Proc Natl Acad Sci USA 89:8794–8797PubMedCrossRefGoogle Scholar
  29. Speicher MR, Ballard G, Ward DC (1996) Karyotyping human chromosomes by combinatorial multi-fluor FISH. Nat Genet 12:368–375PubMedCrossRefGoogle Scholar
  30. Srivastava AK, Hagino Y, Schlessinger D, et al. (1993) Ribosomal DNA clusters in pulsed-field gel electrophoretic analysis of human acrocentric chromosomes. Mammal Genome 4:445–450CrossRefGoogle Scholar
  31. Torchia BS, Call LM, Migeon BR (1994) DNA replication analysis of FMRl, XIST, and factor 8C loci by FISH shows nontranscribed X-linked genes replicate late. Am J Hum Genet 55:96–104PubMedGoogle Scholar
  32. Trask BJ (1991) Fluorescence in situ hybridization: applications in cytogenetics and gene mapping. Trends Genet 7:149–154PubMedGoogle Scholar
  33. Weier H-UG, Wang M, Mulliken JC, et al. (1995) Quantitative DNA fiber mapping. Hum Mol Genet 4:1903–1910PubMedCrossRefGoogle Scholar
  34. Woodward K, Kendall E, Vetrie D, et al. (1998) Pelizaeus-Merzbacher disease: identification of Xq22 proteolipid-protein duplications and characterization of breakpoints by interphase FISH. Am J Hum Genet 63:207–217PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2001

Authors and Affiliations

  • Orlando J. Miller
    • 1
  • Eeva Therman
    • 2
  1. 1.Center for Molecular Medicine and GeneticsWayne State University School of MedicineDetroitUSA
  2. 2.Laboratory of GeneticsUniversity of WisconsinMadisonUSA

Personalised recommendations