2] High-efficiency full-length cDNA cloning

Abstract

Publisher Summary A full-length cDNA library is advantageous in that it allows cloning of a complete sequence in a single step. However, the representation of full-length cDNA clones has been low in cDNA libraries prepared using standard techniques. In the preparation of full-length cDNA libraries, two problems have arisen. The first is the difficulty in reaching the cap site in the first-strand synthesis with the reverse transcriptase (RT), the first enzyme involved in the preparation of a cDNA library. The other problem in library preparation is that there have not been effective methods for selection of full-length cDNAs from incompletely extended cDNAs. This chapter describes protocols using the powerful “thermostabilized” RT that make it possible to prepare efficiently full-length cDNAs longer than 10 kb, combined with the selection of cDNA by biotinylated cap trapper to remove residual non-full-length cDNAs. Using these protocols, full-length cDNA libraries can be prepared at high yield without using polymerase chain reaction (PCR) that introduces sequence bias and causes overrepresentation of short clones in a library.