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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 int…
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.
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Q1: Why is RACE used to obtain full-length cDNA sequences?
RACE is used because novel mRNA transcripts often yield partial cDNAs with unknown sequences at the 5' or 3' ends. This technique extends partial cDNA sequences from a known internal site to the unknown ends using anchored PCR, enabling researchers to obtain complete cDNA for the transcript.
Q2: How does the hybrid primer function in 3' RACE?
The hybrid primer consists of an oligo-dT sequence linked to a unique anchor sequence. The oligo-dT binds the poly-A tail at the 3' end of mature mRNAs, while the anchor adds unique nucleotides upstream of the poly-T sequence in the newly synthesized first cDNA strand.
Q3: What is the role of nested primers in RACE amplification?
Nested primers bind downstream of primers from the first PCR round, increasing specificity and yield of full-length cDNAs. Since adapter primers can amplify off-target sequences, a second amplification cycle with nested primers reduces non-specific products and improves the recovery of the desired cDNA.
Q4: How does 5' RACE differ from 3' RACE in obtaining unknown sequences?
In 5' RACE, a gene-specific primer binds the 3' end of mRNA during reverse transcription, then terminal deoxynucleotidyl transferase adds a poly-A tail to the cDNA's 3' end. An adapter primer with oligo-dT and anchor sequences then synthesizes the complementary strand to amplify the missing 5' sequence.
Q5: What are degenerate primers and why can they cause problems in RACE?
Degenerate primers are designed as possible cDNA end sequences predicted from amino acid sequences of encoded proteins. Since amino acids are coded by multiple mRNA codons, predicting nucleotide sequences from protein sequences is inaccurate, leading degenerate primers to produce undesirable PCR products.
Q6: How do GI tail primers improve RACE specificity compared to G-rich primers?
GI tail primers contain a mixture of guanines and inosine, increasing PCR specificity and reducing off-target amplification. Unlike G-rich primers that require substantially higher annealing temperatures, GI tail primers anneal at standard PCR temperatures, making them more practical for routine RACE applications.
Q7: What modifications enhance RACE technique performance for obtaining complete cDNA?
RACE modifications include optimizing anchor sequences, tailoring primer sequences to specific cDNAs, using degenerate primers, controlling GC composition, adjusting cDNA end length, and employing poly(C) tails with GI tail hybrid primers. These improvements increase specificity and yield of full-length cDNA sequences through next generation sequencing reversible terminator approaches.