Understanding RACE PCR - (Mar/31/2013 )
I'm trying to understand this Nature protocol for classic 3' RACE PCR:
One thing I don't seem to understand is the design of the 3' end primer. In the paper, Qt=Qo+Qi+TTTTTTTTTTTTTTT
I understand that Qi contains the restriction sites for Hind III, Sst I and Xho I. Q outer (Qo) is 5'–ccagtgagcagagtgacg–3'. I simply don't understand what Qo is used for and why Qo has this sequence. The protocol says "The end is amplified using a primer that contains part of thissequence (Qouter, QO) and that binds to these cDNAs at their 3' ends ends, and a primer that matches the gene of interest (gene-specific primer 1; GSP1).
Can someone please help clarify why Qo has the sequence is does, and what it is exactly used for? I'm still not clear on why it is in the primer. Thanks!
Usually RACE primers contain something that anneals to the cDNA in the first round (in this case the oligo T sequence, for the polyA tail) and then some specific sequence, that is used for primer annealing in next round.
This primer would anneal to all polyA containing sequences = all mRNA. Your GSP1 only matches yours gene mRNA. Since you need a pair of primers to amplify, GSP1 and the Qo sequence is used to amplify sequence between GSP and the 3' end of your gene. Qo (or probably Qo+Qi, not read it) is a second primer, it's usually a specifically designed sequence, that doesn't have any sequence similarity to any existing gene, so it only amplifies the Qt primers that annealed on 3' ends.
That is to increase the specificity in later cycles and because obviously TTTTTTTT... sequence is not a good primer for PCR itself.