Constructing and screening cDNA libraries

Abstract

The in-depth study of eukaryotic gene structure and expression has been made possible by the ability to clone transcribed DNA. Since RNA per se cannot be cloned (i.e. stably introduced and replicated in a bacterial cell) and RNA-DNA hybrids can only be cloned with very low efficiency, the best choice for the isolation of mRNA is its conversion into double-stranded cDNA. Thus, a good strategy for the isolation of a particular eukaryotic gene is the screening of a cDNA library representing the mRNA population of the appropriate tissue or cell type using an available nucleic acid as a probe. Alternatively, the clone of interest can be identified by virtue of the expression of a specific protein by the target mRNA. In the first case, the hybridization probe could be an existing cloned gene from the same or another species, an oligonucleotide or a pool of oligonucleotides (Suggs et al., 1981), or a PCR product (Lee et al., 1988). In the second approach expressed proteins can be detected by specific antibodies (Young and Davis, 1983b) or by functional assays that determine their biological activity (Yang et al., 1986).