Nucleic Acid Hybridization

Abstract

Nucleic acid hybridization allows the identification of homologous DNA and RNA sequences. This technique is used for gene mapping, gene expression studies and analysis of genomic organization.

Keywords: deoxyribonucleic acid; ribonucleic acid; hybridization; probe; Southern

Figure 1.

Southern blotting. (a) Southern blotting procedure. (i) digested DNA is separated by electrophoresis on an agarose gel; (ii) the gel is treated with chemicals to denature the DNA; (iii) the DNA is transferred to a membrane (see (b)), retaining the pattern of fragments; (iv) the membrane is incubated with hybridization solution containing radioactively labeled probe DNA, the probe anneals to complementary DNA sequences on the membrane; (v) excess probe is removed by washing and the membrane is exposed to autoradiographic film; (vi) the radioactive probe produces signals on the autoradiograph corresponding to the position of the DNA on the membrane. (b) Schematic diagram of blotting apparatus. (i) Upward capillary transfer. The gel is placed on blotting paper soaked in transfer solution which acts as a wick, supported by a glass plate. The DNA is transferred upwards to the membrane via capillary action as the solution is drawn up through a stack of blotting papers on top of the membrane. The apparatus is weighted to promote even transfer. (ii) Downward capillary transfer. This method prevents compression of the gel, and may result in more rapid and efficient transfer.

Figure 2.

(a) Autoradiograph from Southern blot. A bacterial artificial chromosome containing mouse DNA sequences was digested with a variety of restriction endonucleases (lanes 1–11) and separated by gel electrophoresis. The DNA was transferred to a nylon membrane, which was probed with a radiolabeled fragment corresponding to a specific gene. (b) Autoradiograph of dot blot. DNA from a human cell line transfected with a plasmid construct was spotted onto a filter. The filter was hybridized with a radiolabeled probe to detect uptake of the construct, with the intensity of the signal in each spot indicating the strength of hybridization. DNA from a cell line known to contain the construct was used as a positive control (open arrow) and DNA from the cell line prior to transfection was used as the negative control (closed arrow). (c) Colony hybridization filter. Bacterial colonies transformed with plasmid DNA were grown on an agar plate. The colonies were transferred to a nylon filter, lysed and the DNA denatured. The filter was hybridized with a DIG‐labeled probe and detection was performed using an alkaline phosphatase conjugated antibody with a colorimetric substrate. The chemical reaction produces an insoluble colored compound on the filter; these signals indicate successfully transformed colonies.

Figure 3.

Other hybridization‐based techniques. (a) Fluorescent in situ hybridization. Metaphase spreads were prepared from a mouse cell line and hybridized to a DIG‐labeled probe generated from mouse major satellite DNA. Detection was performed using an antibody conjugated to the fluorophore Texas Red (red), and chromatin counterstained with 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI, blue). Images were captured under a fluorescent microscope. (b) Microarray hybridization. A microarray of BAC DNA was hybridized with two different probes labeled with Cy3 (green) and Cy5 (red) fluorophores (the combined signal from the probes appears yellow). The level of signal from each probe on any spot indicates the degree of homology with the probe, and the difference can be accurately quantitated.

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References

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Further Reading

Beck S and Koster H (1990) Applications of dioxetane chemiluminescent probes to molecular biology. Analytical Chemistry 62: 2258–2270.

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

Choo KHA, Craig JM, Cutts SM and Lo AWI (1999) In situ hybridization. Encyclopedia of Life Sciences, vol. 10, pp. 161–165, http://www.els.net. London, UK: Nature Publishing Group.

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Khan J, Bittner M, Chen Y, Meltzer P and Trent JM (1999) DNA microarray technology: the anticipated impact on the study of human disease. Biochimica et Biophysica Acta 1423: M17–M28.

Maniatis T, Fritsch EF and Sambrook J (1986) Molecular Cloning – A Laboratory Manual. New York, NY: Cold Spring Harbor Laboratory Press.

Wetmur JG (1976) Hybridization and renaturation kinetics of nucleic acids. Annual Review in Biophysics and Bioengineering 5: 337–361.

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How to Cite close
Chan, Sarah WL, and Choo, Andy KH(Sep 2005) Nucleic Acid Hybridization. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0005338]