Subtraction hybridization employing aziridinylbenoquinone cross-linking agents
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United States of America Patent
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Jan 7, 1997
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app pub date -
Apr 7, 1993
filing date -
Apr 7, 1993
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Abstract
A novel process is disclosed utilizing a subtraction hybridization technique for producing DNA hybridization probes of high specific activity or cDNA subtraction libraries, useful in connection with the cloning of differentially expressed genes. A preparation of single stranded cDNA derived from transcript mRNA of a target cell source is subjected to subtraction hybridization using excess single stranded 'driver' nucleic acid from a reference cell source so that all the cDNA having a nucleotide sequence complementary to transcript mRNA of the reference cell source is subtracted by annealing with the 'driver' nucleic acid to form duplex molecules. The strands of these duplex molecules are then chemically cross-linked by treatment with an aziridinylbenzoquinone interstrand cross-linking agent, and the remaining unsubtracted single stranded unique cDNA derived solely from the target cell source is processed in the presence of the chemically cross-linked duplex molecules to provide labelled probe material or a subtraction cDNA library, using random priming and a DNA polymerase lacking exonuclease activity and inactive with respect to the cross-linked duplex molecules.
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