Direct cloning of differential expression genes with full-length by a modified solid subtractive hybridization

A simple and efficient cDNA subtraction technique was developed based on solid-support magnetized beads and polymerase chain reactions to identify the differential expression genes.This strategy is efficient,easy to use and the whole process including mRNA isolation,cDNA synthesis and subtractive hybridization is performed on magnetized beads.It can be applied to any comparative studies on gene expression in different populations.Driver mRNA isolation and first-strand cDNA synthesis were performed by biotinylated oligo(dT) primer on magnetized beads.Tester mRNA isolation,double-strand cDNA synthesis and adapter ligation were also performed by biotinylated oligo(dT) primer on magnetized beads. Then the second strand was eluted and subtracted by first strand of driver cDNA hung on the beads for the efficient removal of common cDNAs.Differentially expressed Tester cDNA was specifically amplified with adapter primer and oligo(dT) primer and then cloned.Dot blotting confirmed that randomly selected clones were differential transcripts of Tester.In the present study,some differentially expressed genes,including several full-length cDNAs,were isolated and screened from coldacclimated Ammopiptanthus mongolicus seedlings by this modified method.Sequence analysis showed that the screened cDNAs shared homologies with previous reported genes involved in plant cold adaptation.These results demonstrate that the modified technique may be of potential value for the isolation of cDNAs expressed in the target tissues without any prior knowledge of the genes.