In Situ Hybridization


In situ hybridization is based on the sequence‐specific annealing of denatured nucleic acid strands. It combines the detection of specific nucleic acid sequences and their localization in relation to cellular or subcellular morphology.

Keywords: DNA/RNA; chromosome; hybridization; genetic diseases; cancer; diagnosis

Figure 1.

The in situ hybridization procedure. (a) Fixation of cellular material (in this case metaphase chromosomes) onto a glass slide. (b) Denaturation of slide‐bound nucleic acid and application of a labelled nucleic acid probe. (c) Hybridization of probe with complementary sequence and direct microscopic visualization of its cytogenetic location.

Figure 2.

Examples of FISH applications where FISH images are obtained by digital capture. (a) Hybridization of normal human metaphase chromosomes (blue) using a centromeric α‐satellite DNA probe (green) at (i) high stringency, showing specific centromeric signals on the cognate chromosomes 13 and 21, and (ii) low stringency, showing a generalized hybridization to all related centromeric α‐satellite on every chromosome. (b) Multicolour (spectral) karyotyping, using differently coloured whole chromosome ‘paints’ on (i) a normal human metaphase spread and (ii) a metaphase spread from a bladder cancer cell line showing multiple chromosome rearrangements (arrows). (Pictures courtesy of Hesed Padilla‐Nash and Thomas Reid). (c) Trisomy detection. (i) Hybridization of a single‐copy chromosome‐21 ‘YAC’ probe (yellow) to a Down syndrome metaphase chromosome spread showing specific mapping of the probe to band q22 (arrows) and the detection of three signals on interphase nuclei. (ii) Detection of three signals (green) on interphase nuclei using a chromosome 18‐specific α‐satellite probe at high stringency, corresponding to a diagnosis of trisomy 18.

Figure 3.

Simultaneous DNA and protein detection. (a) A composite digital image for FISH using an α‐satellite probe (green) specific for chromosomes 13 and 21 and the immunocytochemical fluorescence detection of a centromere‐specific protein (pink) to demonstrate colocalization of the centromere protein and the DNA probe. (b) and (c) Using digital technology, the composite image can be split into its colour constituents to show signals for the chromosome 13‐ and 21‐specific α‐satellite sequences and the centromere protein, respectively.


Further Reading

Bickmore WA (ed.) (1999) Chromosome Analysis: a Practical Approach. Oxford: Oxford University Press.

Choo KHA (ed.) (1994) In Situ Hybridization Protocols. Totowa, NJ: Humana Press.

du Sart D and Choo KHA (1996) The technique of in situ hybridization: principles and applications. In: Repley R and Walker JM (eds) Molecular Biomethods Handbook, pp. 697–720. Totowa, NJ: Humana Press.

Gole LA and Bongso A (1997) Fluorescence in situ hybridization – some of its applications in clinical cytogenetics. Singapore Medical Journal 38: 497–503.

Heng HHQ, Spyropoulos B and Moens PB (1997) FISH technology in chromosome and genome research. BioEssays 19: 75–84.

Lichter P (1997) Multicolour FISHing: what's the catch? Trends in Genetics 13: 475–479.

McNicol AM and Farquharson MA (1997) In situ hybridization and its diagnostic applications in pathology. Journal of Pathology 182: 250–261.

Raap AK (1998) Advances in in situ hybridization. Mutation Research 400: 287–298.

Ramsay G (1998) DNA chips: state‐of‐the art. Nature Biotechnology 16: 40–44.

Wienberg J and Stanyon R (1995) Chromosome painting in mammals as an approach to comparative genomics. Current Opinion in Genetics and Development 5: 792–797.

Wilkinson DG (ed.) (1999) In Situ Hybridization: A Practical Approach, 2nd edn. Oxford: Oxford University Press.

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Choo, KH Andy, Craig, Jeffrey M, Cutts, Suzanne M, and Lo, Anthony WI(Apr 2001) In Situ Hybridization. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0002646]