15.11: RACE - Rapid Amplification of cDNA Ends
Rapid Amplification of cDNA Ends or RACE is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
Since the invention of RACE, the technique has continuously been modified to increase the specificity and yield, such as improvising the anchor sequences or optimizing the primer sequences, often tailored to the cDNA that requires amplification.
One alteration uses a degenerate primer instead of the gene-specific primer derived from the known cDNA sequence. A degenerate primer is designed as a possible sequence of the cDNA end, predicted from the amino acid sequence of the encoded protein. Because an amino acid can be coded by more than one mRNA codon, predicting the nucleotide sequence from a protein sequence is not always the most accurate. As a result, degenerate primers are known to lead to undesirable PCR products. The specificity of the reaction can be controlled by optimizing the GC composition of the primer or controlling the length of the cDNA ends.
Another approach has been to add a poly(C) tail to the 5’ end instead of a poly(A) tail. The template is then amplified using a hybrid primer with a poly(G) tail or a mixture of G's and inosine (GI tail). Such a specific hybrid primer increases the specificity of the PCR, reducing off-target amplification. The GI tail can anneal at temperatures normally used in PCR, as opposed to a G-rich primer that requires substantially higher annealing temperatures.