Directional cloning of cDNA using a selectable SfiI cassette

Abstract

To increase the efficiency of directionally cloning cDNA, we have constructed a pair of vectors and devised cDNA cloning strategy that improves upon previously published methods. The vectors, pLIB: AZ and pLIB: ZA, have to unique (distinct religation specificities; GGCCN/NNNNGGCC) SfiI sites (Sfil.A and Sfil.b) flanking a stuffer fragment which contains the tetracycline-resistance element. These vectors permit the directional cloning of cDNA in both sense (pLIB:AZ) and antisense (pLIB:ZA) orientation srelative to the promoter for phage T3 RNA polymerase. cDNA that was synthesized using a primer with a 5′ sequence of a Sfil.B site followed by an oligo(dT) 163′ tail was then ligated to an adaptor with the sequence of a Sfil.A site produced directional molecules that could be cloned into the pLIB vectors. Complex libraries with 10 7 members were produced from as few as 6 × 10 5 cells. The SfiI sites and suffer can be subcloned as a cassette to permit directional cloning in other vectors, as there are several restriction enzyme sites flanking this region to the 5′ and 3′.