Rapid amplification of cDNA ends


Rapid amplification of cDNA ends (commonly abbreviated to RACE; also one-sided PCR or anchored PCR) is a technique used in transcript analysis to obtain the full sequence of a messenger RNA. The RNA of interest is reverse-transcribed to single-stranded cDNA, which is then amplified in a PCR fashion. The primer used to synthesise cDNA is called a gene-specific primer; it hybridises to a known sequence within the mRNA and is extended towards an unknown sequence at either end (either 5' RACE or 3' RACE).

5' RACE is useful in identifying the transcription start site on mRNA.

1. First, an anti-sense oligonucleotide called the gene specific primer is hybridised to a known sequence in the mRNA of interest.
2. The primer is then extended by reverse transcriptase, using mRNA as a template, to make a single stranded cDNA copy.
3. The enzyme terminal deoxynucleotidyl transferase (TdT) is used to add a string of identical nucleotides to the 3' end of the nascent cDNA, called the homopolymeric tail. 4. The modified cDNA is then used as a template for PCR by annealing a sense, universal primer that can anneal to the homopolymeric tail on the 3' end of the cDNA. The cDNA template can be made double-stranded in this way.
5. Once amplified, the cDNA is cloned into a vector and sequenced. The first base in the sequence that is not a complement of the 3' homopolymeric tail marks the start of the RNA transcript.