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subtractive DNA cloning

A difference cloning method; a technique for comparison of two closely related cell types (e.g. a cell with a deletion mutation and the normal cell), and isolation from one (the tester) of DNA that is absent from the other (the driver). tester DNA is cleaved with a restriction nuclease that will allow its subsequent insertion into a cloning vector; an excess of driver DNA is randomly sheared. The two preparations are mixed, melted and annealed to form, among other species, duplexes composed of strands of the different sources (inter-resource duplex, IRD). The fraction of the annealed product, both strands of which were cleaved at restriction sites and which therefore is capable of being cloned, will be enriched in those sequences for which there was no competing randomly sheared driver DNA, i.e. those that are unique to the tester DNA, especially when annealing is terminated before an equilibrium is established (kinetic enrichment). The unique DNA sequences will therefore form a high proportion of the successfully cloned annealed DNA product. (see also magnet-assisted subtractive technique (MAST); subtractive hybridization)

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