Rapid Amplification of Sequences from the 3′ Ends of mRNAs: 3′-RACE

Abstract

cDNAs whose first-strand synthesis has been primed by oligo(dT) generally contain the 3′ sequences of the RNA. However, cDNAs generated by internal gene-specific primers will usually lack one or both terminal regions of the parental mRNA. And alternate splicing or the presence of an internal A-rich tract can generate cDNA clones that lack 3′ sequences. These sequences can be recovered using 3′-RACE (rapid amplification of cDNA ends), which can also be used to map the 3′ termini of families of mRNAs with alternative polyadenylation sites. Briefly, a population of mRNAs is transcribed into cDNA using an adaptor–primer with a poly(T) at its 3′ end and 30–40 nt containing the recognition sites for two or three restriction enzymes at its 5′ end. Reverse transcription is usually followed by two successive rounds of polymerase chain reaction (PCR). The first is primed by a gene-specific sense oligonucleotide and the antisense (dT) adaptor–primer. The products of the first PCR can be used as templates for a second, nested PCR, which is primed by a gene-specific sense oligonucleotide internal to the first, and a second antisense oligonucleotide whose sequence is identical to the central region of the (dT) adaptor–primer. The products amplified in the second PCR are isolated, cleaved with a suitable restriction enzyme, cloned, and characterized.