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Nucleic Acids Research, 1994, Vol. 22, No. 21 4381-4385
© 1994

METHODS

Differential cDNA cloning by enzymatic degrading subtraction (EDS)

Jin Zeng*, Roger A. Gorski1 and Dean Hamer

Laboratory of Biochemistry, National cancer Institute, National Institutes of Health Building 37, Room 4A13, Bethesda, MD 20893 1Department of Anatomy and Cell Biology, UCLA school of Medicine, Center for Health Sciences Los Angeles, CA 90024, USA

*To whom correspondence should be addressed

Received August 2, 1994. Revised September 21, 1994. Accepted September 21, 1994.

We describe a new method, called enzymatic degrading subtraction (EDS), for the construction of subtractive libraries from PCR amplified cDNA. The novel features of this method are that i) the tester DNA is blocked by thionucleotide incorporation; (ii) the rate of hybridization is accelerated by phenol-emulsion reassociation; and (iii) the driver cDNA and hybrid molecules are enzymatically removed by digestion with exonucleases III and VII rather than by physical partitioning. We demonstrate the utility of EDS by constructing a subtractive library enriched for cDNAs expressed in adult but not in embryonic rat brains.


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