15.11 : RACE - Amplificação Rápida de Terminações de cDNA

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.