- This article is about the molecular biology technique, for alternate meanings, see RACE
RACE, or Rapid Amplification of cDNA Ends, is a technique used in molecular
biology to amplify the ends of messenger RNA (mRNA) transcripts using a specialized
reverse transcription-polymerase chain reaction (RT-PCR). It allows the amplification of an unknown end portion of a
transcript using known information from the center of the transcript. It can be used to obtain the 5' end (5' RACE-PCR) or 3'
end (3' RACE-PCR) of mRNA. This technique is sometimes called one-sided PCR or anchored PCR.
The protocols for 5' or 3' RACE differ slightly. 5' RACE-PCR begins using mRNA as a template for a first round of cDNA synthesis (or reverse transcription) reaction using an anti-sense (reverse) oligonucleotide primer specific that recognizes known sequence in the gene of interest; the primer is called a gene specific primer (GSP) and copies the mRNA template in the 3' to the 5' direction to generate a specific single stranded cDNA product. Following first strand cDNA synthesis, the enzyme terminal deoxynucleotidyl transferase (TdT) is used to add a homopolymeric tail (i.e. a string of identical nucleotides) to the 5' end of the cDNA. A PCR reaction is then carried out, which uses a second anti-sense gene specific primer (GSP2) that binds to the known sequence, and a sense (forward) general universal primer (UP) that binds the homopolymeric tail added to the 5' 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 mRNAs for priming during reverse transcription, so this method does not require the addition of nucleotides by TdT. First strand cDNAs are generated using an Oligo-dT-adaptor primer that complements the polyA stretch and adds a special adaptor sequence to the 3' end of each cDNA. PCR is then used to amplify 3' cDNA from a known region in the specific cDNA using a sense GSP, and an anti-sense primer complementary to the added adaptor sequence.
Source: "Rapid amplification of 5' cDNA ends," in Molecular Cloning: A Laboratory Manual (eds. Sambrook, J. & Russell, D.W.) Chapter 8 Protocol 9, 8.54−8.60 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2001)
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