Cloning Full-Length Transcripts and Transcript Variants Using 5′ and 3′ RACE

Abstract

Gene transcripts and transcript variants must be cloned to characterize gene function and regulation. However, obtaining full-length cDNAs with accurate sequences from the 5′ end through to the 3′ end can be challenging. Here we describe a reverse-transcriptase-based method for obtaining full-length cDNAs using the SMARTer (“Switching Mechanism At RNA Termini”) RACE technology developed by Clontech. RNA is isolated from the tissue of interest and annealed to a primer (a modified oligo(dT) primer for polyA+ transcripts; random hexamers or a gene-specific primer for polyA− transcripts). A modified MMLV-reverse transcriptase uses the primer to initiate cDNA synthesis from RNA transcript(s) annealed to the primer and continues cDNA synthesis (reverse transcription) towards the 5′ end of the transcript(s). Importantly, this reverse transcriptase possesses terminal transferase activity, so when it reaches the 5′ end of a transcript it adds a 3–5 residue “tail” to the newly synthesized cDNA strand. Included in the reverse transcriptase reaction mix is an oligonucleotide containing a sequence tag as well as a terminal series of modified bases that anneal to the 3–5 residue tail on the newly synthesized cDNA. The reverse transcriptase proceeds from the end of the transcript onwards into the modified bases and the rest of the sequence-tagged oligo. The newly synthesized cDNA now has a sequence tag attached to it and can be used as a template for PCR, with one primer complementary to the sequence tag and the second primer specific to the gene of interest. The fragment can be cloned and sequenced or just sequenced directly. If high-quality, undegraded RNA is used, obtaining the true 5′ end of a transcript is greatly enhanced. In combination with 3′ RACE, full-length transcripts are easily cloned. This method provides sequence information on important regulatory regions, such as 5′ and 3′ UTRs and flanking regions, and is ideal for detecting transcript variants, including those with alternative transcriptional start sites, alternative splicing, and/or alternative polyadenylation.