Nucleic Acids: Subtractive Hybridization


Cells can be characterized phenotypically by demonstrating expressed proteins or messenger RNA. ‘Subtracting’ the gene expression profile of one cell from that of a different cell eliminates the signal from common shared ‘housekeeping’ genes and allows the functional gene expression profile of one cell to be defined. Subtractive hybridization is one of the major methods for achieving this.

Keywords: subtraction; hybridization; phenotyping; poly(A) PCR; mRNA

Figure 1.

Principle of subtractive hybridization. Driver cDNA is photobiotinylated prior to use and hybridized in a 20‐fold excess with tracer cDNA. Biotinylated driver/tracer hybrids and unhybridized driver cDNA species are removed by streptavidin precipitation of biotin, leaving the first subtraction product, S1. This is then substituted for the tracer cDNA in a repeat of the process, yielding a second subtraction product, S2. Two further rounds of subtraction, using S2 and its product S3 in the place of tracer cDNA, yield the final subtraction product, S4, which comprises cDNA representative of all the mRNA present in the tracer, but not the driver cDNA.



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Further Reading

Brady G, Billia F and Knox J et al. (1995) Analysis of gene expression in a complex differentiation hierarchy by global amplification of cDNA from single cells. Current Biology 5: 909–922.

Byers RJ, Hoyland JA, Dixon J and Freemont AJ (2000) Subtractive hybridization – genetic takeaways and the search for meaning. International Journal of Experimental Pathology 81: 391–404.

Coloantuoni C, Purcell AE, Bouton CML and Pevsner J (2000) High throughput analysis of gene expression in the human brain. Journal of Neuroscience Research 59: 1–10.

Hoyland JA, Byers RJ and Freemont AJ (2001) New technologies for detecting alterations in genes and gene expression. In: Lowe DG and Underwood JCE (eds) Recent Advances in Histopathology 19, pp. 181–200. Edinburgh: Churchill Livingstone.

Peale FV and Gerritsen ME (2001) Gene profiling techniques and their application in angiogenesis and vascular development. Journal of Pathology 195: 7–19.

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Byers, Richard J, Hoyland, Judith A, and Freemont, Anthony J(May 2005) Nucleic Acids: Subtractive Hybridization. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1038/npg.els.0003149]