AN IMPROVED METHOD FOR SUBTRACTIVE CLONING OF DIFFERENTIALLY EXPRESSED GENES IN HIGHER-PLANTS BY PROTECTIVE EXONUCLEASE DIGESTION AND DISCRIMINATING PCR AMPLIFICATION

An improved method for subtractive cloning with enhanced efficiency wa s developed by modifying the enzymatic degrading subtraction. The thio nucleotide-modified tester cDNA fragments under control of one linker- primer were hybridized with excess driver cDNA fragments flanked by th e other distinct linker-primer. After selective digestion of incomplet ely protected tester/driver and of unprotected driver/driver molecules with exonuclease III and VII, the protected tester/tester reassociate s due to thionucleotides were exclusively amplified by PCR with the te ster-cDNA-specific primer. The subtractively enriched target cDNA frag ments, showing distinct bands in an agarose gel, were inserted into pU C19, and random colonies with inserts were screened by Northern hybrid ization to tester and driver RNA. Four distinct clones were confirmed to be up-regulated by the withdrawal of potassium from the nutrient so lution of seedling barley growing hydroponically. The original protoco l generated only smeared amplicons due to non-selective PCR amplificat ion of the hybridized cDNA mixture including remains of undigested dri ver cDNA.