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 was developed by modifying the enzymatic degrading subtraction. The thionucleotide-modified tester cDNA fragments under control of one linker-primer were hybridized with excess driver cDNA fragments flanked by the other distinct linker-primer. After selective digestion of incompletely protected tester/driver and of unprotected driver/driver molecules with exonuclease III and VII, the protected tester/tester reassociates due to thionucleotides were exclusively amplified by PCR with the tester-cDNA-specific primer. The subtractively enriched target cDNA fragments, showing distinct bands in an agarose gel, were inserted into pUC19, and random colonies with inserts were screened by Northern hybridization to tester and driver RNA. Four distinct clones were confirmed to be up-regulated by the withdrawal of potassium from the nutrient solution of seedling barley growing hydroponically. The original protocol generated only smeared amplicons due to non-selective PCR amplification of the hybridized cDNA mixture including remains of undigested driver cDNA.