Fig. 1
Schematic drawings of DSH and construction of the cDNA library of upregulated transcripts. a Different primers were used for the synthesis of first-strand tester and driver cDNAs. White and black boxes represent the 5′ and 3′ ends of the tester-cDNAs and the attB1 and attB2 sites in primers attB1-TSO and attB2-TSpolyT30, respectively. Striped boxes represent the 5′ and 3′ ends of driver-cDNAs and corresponding primers 5’CPEx and 3’APEx, respectively. Red boxes represent the 5′ ends of the tester and corresponding primer tester-enrichF. Blue boxes represent the SfiI sites and, together with yellow boxes, represent the 3′ ends of tester and corresponding primer tester-enrichR. Solid lines represent constitutive cDNAs. Dashed lines represent upregulated cDNAs. Primers tester-enrichF/tester-enrichR were used to enrich tester. 5’CPEx/3’APEx were used for driver enrichment. The excessive driver was hybridized with tester and renatured double-strand cDNAs were removed by the DSN. The resulting single-strand molecules, mostly upregulated cDNAs, can be amplified by tester-enrichF/tester-enrichR. b Flow chart of construction of the library of upregulated cDNAs. The SfiI-digested cDNA molecules were ligated with left and right arms of the phage vector λTriplEx2. The ligation product was packaged and used for transformation of E.coli strain BM25.8 to obtain the plasmid cDNA library in pTriplEx2. The resulting products of SfiI-digested pTriplEx2 library were size fractionated with agarose gel electrophoresis, and different sized fragments were used for the construction of cDNA expression libraries via the Gateway® cloning technique