Screening Recombinant Libraries by Polymerase Chain Reaction

The complexity of the genome of a particular organism or the relative abundance of a particular mRNA, within the cell type from which a cDNA library was constructed, affects the ability with which one can isolate a gene or cDNA clone of interest. With respect to genomic libraries, the number of clones needed to be screened to isolate a single-copy sequence is a function of the complexity of the genome and the average size of the cloned fragments in the library (1 ). In the case of cDNA libraries, the frequency of a given clone of interest depends on the abundance of the messenger RNA. Highly abundant messages can represent 10% or more of total mRNA, whereas very rare messages can be as low as one in 10⁶ . In addition, the representation of some sequences in a cDNA library, particularly the 5′ ends of large mRNAs, will be less than expected due to the technical difficulties in converting the mRNA into full-length cDNA copies. In some cases, a particular sequence of interest can be depleted or lost at various steps of screening due to its inefficiency to be replicated relative to other clones in the library.