Rapid Amplification of Sequences from the 5′ Ends of mRNAs: 5′-RACE

Isolating a full-length clone of cDNA provides certainty that the entire protein-coding sequence of the mRNA has been identified and allows the 5' end of the mRNA to be precisely mapped onto the genomic DNA sequence. Unfortunately, partial clones that lack sequences corresponding to the 5' end of the target mRNA occur commonly in cDNA libraries. Rapid amplification of cDNA ends (RACE) provides a means to overcome this obstacle by amplifying the 5'-terminal sequences of cDNA. This technique differs from conventional polymerase chain reaction in that it only requires knowledge of a small region of sequence within either the target RNA or in a partial clone of cDNA. In the first step of 5'-RACE, extension of the primer by reverse transcriptase yields single-stranded cDNAs complementary to the 5' regions of the mRNA. In the second step, a homopolymeric tail or a primer-adaptor is added to the 3' ends of the cDNAs. This generates a primer-binding site upstream of the unknown 5'-sequence of the target mRNA. Finally, synthesis of the second cDNA strand and amplification of the resulting double-stranded cDNAs is performed using the gene-specific primer and the upstream primer. The double-stranded products of the final stage are purified and cloned into a vector for sequencing analysis and subsequent manipulation.