An RT-PCR-Based Protocol for the Rapid Generation of Large, Representative cDNA Libraries for Expression Screening

The ability to isolate genes that encode specific proteins has been fundamental to the revolution in molecular and cell biology over the past two decades. The protein-encoding versions of genes are isolated from cDNA libraries. A cDNA library is constructed by isolating mRNA from the cell or tissue that expresses the target protein, converting all the mRNA into cDNA copies via the enzyme RT, and cloning all the cDNAs into bacterial cells via a plasmid or bacteriophage vector. The library is then screened with a probe to detect a clone expressing the protein of interest. The probe is usually either a mixed oligonucleotide probe based on partial amino-acid sequence data for the protein, or alternatively, an antibody specific for the protein of interest. For the latter approach, an expression vector must be used to enable the cloned cDNA to be expressed at the protein level in E. coli. Advances in the methodologies involved in cDNA cloning during the 1980’s enabled the identification and characterization of the cDNAs encoding thousands of important human proteins. With the advent of PCR, an alternative approach to cDNA isolation has also been employed. By using degenerate primers based on partial amino-acid sequence information from the protein of interest, its cDNA may be amplified from total cDNA without the need for cDNA library construction and screening. cDNA libraries are still valuable tools when full-length clones are sought, and in particular, when amino-acid sequence information is unavailable.