Construction of directional cDNA libraries enriched for full-length inserts in a transcription-competent vector

Abstract

We have designed a simple procedure for the construction of directional cDNA libraries enriched for full-length inserts in a transcription-competent cloning vector. An oligonucleotide, its 5′ end starting with a heteropolymeric sequence encoding the rare restriction sites for NotI and SfiI, followed by 50 dT residues, is used to prime first-strand synthesis on size-selected mRNA. After second-strand synthesis and EcoRI linker addition, the cDNA is double digested with EcoRI and NotI, or with EcoRI and SfiI, to generate DNA fragments with asymmetric ends that can be directionally cloned. The cDNA fragments are enriched for “full length” by size selection and ligated into a phage lambda vector containing the T3 and T7 RNA polymerase promotors. These cDNA libraries can directly be used for in vitro synthesis of sense or antisense RNA.