Isolation of full-length gene transcripts is important to determine the protein coding region and study gene structure. However, isolation of novel gene sequences is often limited to expressed sequence tags (ESTs) (i.e., short cDNA fragments that predominantly represent the 3′ end of the transcript). Rapid amplification of cDNA ends (RACE) is today by far the most popular approach for obtaining full-length cDNAs when only part of the transcript's sequence is known.

Since its original description (1,2) numerous modifications and improvements of the method have been developed and consist of a collection of PCR-based cloning procedures that extend a known cDNA fragment toward the 3′ (3′ RACE) or the 5′ (5′ RACE) cDNA end. The original method is based on attachment of an anchor sequence to one end of the cDNA that can be used as a primer binding template in PCR with a second gene-specific primer from the known part of the gene. Although this procedure seems in theory fast and simple, it is technically difficult and usually requires substantial optimization and several repetitions before satisfactory results can be obtained (3). This is particularly due to the use of a universal primer corresponding to the anchor sequence present in all cDNAs, which may result in a high background of nonspecific products even after a nested PCR with a gene-specific primer internal to the first gene-specific primer is performed. Another drawback of the method is the difficulty of obtaining the full-length 5′ end of the transcript due to the presence of many truncated transcripts in the messenger RNA (mRNA) pool. Several strategies aimed at eliminating these problems have been developed (4,5,6,7,8,9) and have proven to be very useful in certain applications. One improvement is based on the utilization of a pair of gene-specific primers in inverse PCR on circularized cDNA templates, which would avoid the use of a universal primer and the problems it may generate (4,6,8,9,10). This strategy also allows the simultaneous isolation of both cDNA ends in a single reaction (9,10). Some of these procedures require the generation of double-stranded cDNA, including the use of template-switching reverse transcription (9) or a post-reverse transcription adaptor ligation step (10). Methods that are performed directly on first-strand cDNA are complicated by the low efficiency of RNA ligase for the circularization reaction (6) or the need for bridging oligonucleotides for this step (8). Furthermore, existing inverse-RACE methods typically require nested PCR to amplify the transcript of interest, and only a limited number of transcripts can be isolated from a single reverse transcription reaction, making it difficult to analyze rare transcripts from scarce tissue.

We describe here an improved inverse-RACE method, which uses CircLigase™ (Epicentre Biotechnologies, Madison, WI, USA) for cDNA circularization, followed by rolling circle amplification (RCA) of the circular cDNA with Φ29 DNA polymerase (New England Biolabs, Ipswich, MA, USA). In this way, a large amount of the PCR template is produced, allowing the simultaneous isolation of the 3′ and 5′ unknown ends of a virtually unlimited number of transcripts after a single reverse transcription reaction. Figure 1 illustrates this method, named RCA-RACE. The process takes advantage of the properties of CircLigase to circularize single-stranded cDNA molecules via an intramolecular link. This ATP-dependent ligase can circularize single-stranded DNA (ssDNA) templates that have a 5′ -phosphate and a 3′ -hydroxyl group and are longer than 30 nucleo-tides. According to the manufacturer, under standard reaction conditions, the enzyme makes essentially no linear or circular concatemers, since it catalyzes only intramolecular ligations. In addition, although CircLigase is influenced by the ssDNA sequence, high concentrations of the enzyme can effectively circularize difficult templates (www.epibio.com/pdftechlit/222pl085.pdf). The circularized cDNA is then amplified in a RCA reaction using the Φ29 DNA polymerase and random primers (11,12). This would allow the generation of enough template for the cloning of rare transcripts, as well as high-throughput cloning of cDNA ends for large numbers of genes from scarce tissue, which cannot be effectively performed with standard RACE methodologies.

Figure 1. The rolling circle amplification rapid amplification of cDNA ends (RCA-RACE) method (Click to enlarge)