A 3′ race protocol to confirm polyadenylation sites

Abstract

A routine procedure in the study of polyadenylation involves the determinations of the junctions of the mRNA body and the poly(A) tail (the poly(A) site). This is typically accomplished by selectively amplifying the 3′-ends of cDNAs (3′-Rapid Amplifi cation of cDNA Ends, or 3′-RACE), followed by sequencing of individual clones. We have developed a modifi cation of the standard 3′-RACE protocol that couples high specifi city with the ability to characterize thousands (or more) of individual sequences. This protocol may be used for numerous purposes, including the confi rmation of results obtained using high throughput sequencing technologies.