3'RACE and the polyA tail - why only one band? (May/21/2009 )
I've been doing 3'RACE to sequence the end of some candidate transcripts, which has worked fine. My problem is with the theory behind 3'RACE. I'm amplifying the end of the mRNA using an oligo dT reverse primer and a gene specific forward primer. Why am I meant to get only one band when the poly(A) tail can be 200-300 A's long and the oligo dT primer could anneal to any stretch of that?
Many RACE kits use a primer TTTTTTTTTTTTTTTTTTV, just to avoid the problem of binding at arbitrary places in the poly-A tail. The V forces extension from the first base which is NOT a T. I don't know where your primer came from, but it might be of this form.
I used the invitrogen kit. The primer sequence is ggccacgcgtcgactagtacttttttttttttttttt. I may be missing something, but isn't the poly-A tail all A's?