5′ end cDNA amplification using classic RACE

The 5′ ends of transcripts provide important information about transcription initiation sites and the approximate locations of local cis-acting enhancer elements; it is therefore important to establish the 5′ ends with some precision. RACE (rapid amplification of cDNA ends) PCR is useful for quickly obtaining full length cDNAs for mRNAs for which only part of the sequence is known and to identify alternative 5′ or 3′ ends of fully sequenced genes. The method consists of using PCR to amplify, from complex mixtures of cellular mRNA, the regions between the known parts of the sequence and non-specific tags appended to the ends of the cDNA. Whereas the poly(A) tail serves to provide such a tag at the 3′ end of the mRNA, an artificial one needs to be generated at the 5′ end, and various approaches have been described to address this step. The classical scheme for 5′ RACE described here is simple, suffices in many instances in which RACE is needed and can be performed in 1-3 days.