Rapid amplification of cDNAends is a technique used in molecular biology to obtain the full length sequence of an RNA transcript found within a cell. RACE results in the production of a cDNA copy of the RNA sequence of interest, produced through reverse transcription, followed by PCR amplification of the cDNA copies. The amplified cDNA copies are then sequenced and, if long enough, should map to a unique genomic region. RACE is commonly followed up by cloning before sequencing of what was originally individual RNA molecules. A more high-throughput alternative which is useful for identification of novel transcript structures, is to sequence the RACE-products by next generation sequencing technologies.
Process
RACE can provide the sequence of an RNA transcript from a small known sequence within the transcript to the 5' end or 3' end of the RNA. This technique is sometimes called one-sided PCR or anchored PCR. The first step in RACE is to use reverse transcription to produce a cDNA copy of a region of the RNA transcript. In this process, an unknown end portion of a transcript is copied using a known sequence from the center of the transcript. The copied region is bounded by the known sequence, at either the 5' or 3' end. The protocols for 5' or 3' RACES differ slightly. 5' RACE-PCR begins using mRNA as a template for a first round of cDNA synthesis reaction using an anti-senseoligonucleotideprimer that recognizes a known sequence in the middle of the gene of interest; the primer is called a gene specific primer. The primer binds to the mRNA, and the enzymereverse transcriptase adds base pairs to the 3' end of the primer to generate a specific single-stranded cDNA product; this is the reverse complement of the mRNA. Following cDNA synthesis, the enzyme terminal deoxynucleotidyl transferase is used to add a string of identical nucleotides, known as a homopolymeric tail, to the 3' end of the cDNA.. PCR is then carried out, which uses a second anti-sense gene specific primer that binds to the known sequence, and a sense universal primer that binds the homopolymeric tail added to the 3' ends of the cDNAs to amplify a cDNA product from the 5' end. 3' RACE-PCR uses the natural polyA tail that exists at the 3' end of all eukaryotic mRNAs for priming during reverse transcription, so this method does not require the addition of nucleotides by TdT. cDNAs are generated using an Oligo-dT-adaptor primer that complements the polyA stretch and adds a special adaptor sequence to the 5' end of each cDNA. PCR is then used to amplify 3' cDNA from a known region using a sense GSP, and an anti-sense primer complementary to the adaptor sequence.
RACE can be used to amplify unknown 5' or 3' parts of RNA molecules where part of the RNA sequence is known and targeted by a gene-specific primer. Combined with high-throughput sequencing for characterization of these amplified RACE products, it is possible to apply the approach to characterize any types of coding or non-coding RNA-molecules. The idea of combining RACE with high-throughput sequencing was first introduced in 2009 as Deep-RACE to perform mapping of Transcription start sites of 17 genes in a single cell-line. For example, In a study from 2014 to accurately map cleavage sites of target RNA directed by synthetic siRNAs, the approach was first named RACE-seq. Further, the methodology was used to characterize full-length unknown parts of novel transcripts and fusion transcripts in colorectal cancer. In another study aiming to characterize unknown transcript structures of lncRNAs, RACE was used in combination with semi-long 454 sequencing.
