Differential cDNA cloning by enzymatic degrading subtraction (EDS).
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.
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
J
J
Zeng
R A
RA
Gorski
D
D
Hamer
D000818:Animals; D015152:Blotting, Northern; D001923:Brain Chemistry; D003001:Cloning, Molecular / Q000379:methods*; D004256:DNA Polymerase I / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics*; D004655:Emulsions; D005090:Exodeoxyribonucleases / Q000378:metabolism; D015723:Gene Library; D009693:Nucleic Acid Hybridization; D019800:Phenol; D010636:Phenols; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D051381:Rats; D017207:Rats, Sprague-Dawley; D013873:Thionucleotides / Q000378:metabolism
D018076:DNA, Complementary; D004655:Emulsions; D010636:Phenols; D012333:RNA, Messenger; D013873:Thionucleotides; D019800:Phenol; D004256:DNA Polymerase I; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C026870:exodeoxyribonuclease VII