4.11
RACE - Amplification rapide des extrémités de l'ADNc
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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.