A 3' RACE protocol to confirm polyadenylation sites.

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 Amplification of cDNA Ends, or 3'-RACE), followed by sequencing of individual clones. We have developed a modification of the standard 3'-RACE protocol that couples high specificity with the ability to characterize thousands (or more) of individual sequences. This protocol may be used for numerous purposes, including the confirmation of results obtained using high throughput sequencing technologies.