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.

Full-length cDNA is not always obtained when novel mRNA gene transcripts are first discovered. Unknown sequences at the ends of the mRNA template can result in partially cloned cDNAs.

The sequence of these partial cDNAs can be extended by a technique called Rapid Amplification of cDNA Ends or RACE to obtain a complete cDNA for the transcript.

In this technique, anchored PCR is used to clone the missing sequence from the known internal site to the 3' or the 5' -end of the mRNA.

In 3’ RACE, the cDNA is synthesized from the mRNA template using reverse transcriptase and a hybrid primer consisting of an oligo-dT sequence linked to a unique nucleotide sequence, called an anchor.

The oligo-dT binds the poly-A tail found at the 3’ end of mature mRNAs, while the anchor part of the primer adds a unique set of nucleotides upstream of the poly-T sequence in the newly synthesized first cDNA strand.

A gene-specific primer or GSP that can pair with the known end of the cDNA sequence is used to synthesize a second DNA strand. The new DNA is denatured, and an adapter primer, complementary to the anchor sequence, and the GSP  are used in multiple rounds of PCR to fill in and amplify the missing 3’  sequence.

In 5’ RACE, a gene-specific primer binds the 3’ end of the mRNA during reverse transcription. Then, terminal deoxynucleotidyl transferase is used to add a poly-A tail to the 3’ end of the resulting cDNA.

After the reverse transcription,  an adapter primer with oligo dT and anchor sequences is used to synthesize a complementary strand.  Then a gene-specific primer and the adapter primer are used to copy and amplify the complete cDNA sequence.

In both types of RACE, the adapter primer can still bind and amplify off-target cDNAs.

Hence, a second amplification cycle is carried out with nested primers that bind downstream of the primers from the first round of PCR. This increases the yield of the specific full-length cDNAs.