ABSTRACT
The ability to generate complementary DNA (cDNA) libraries is one of the most fundamental procedures of contemporary molecular biology. A major concern of current methods is that the majority of cDNAs present in any given library are incomplete, rendering the characterization of genes an inefficient and time-consuming task. We have developed an affinity selection method to purify mRNAs via non-covalent capture of the 5′ cap structure. This protocol can also be used to enrich reverse transcription reaction products for full-length cDNAs. The key features of this method are a bifunctional fusion protein that can be immobilized onto a solid support matrix and which binds 5′ cap structures of eukaryotic mRNAs. Following first strand cDNA synthesis, a single strand specific nuclease is used to remove cap structures from incomplete mRNA:cDNA duplexes. Specific enrichment of complete mRNA:cDNA duplexes is then achieved using this novel affinity matrix. This method can be used to enrich for full-length cDNAs or cDNA clones having complete 5′ ends and to generate cDNA libraries in which reverse transcribed products of polyadenylated and nonpolyadenylated mRNAs are equally represented.
