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?
With the oligo-T primer anneals to the poly-A tail, a location is selected which will lie somewhere in the poly-A and likely will produce, from that copying reaction, a shorter poly-A. In the next round of amplification, again a oligo-T primer anneals to that new poly-A tail, but this time the poly-A tail going into the reaction is shorter and the poly-A tail produced will likely be shorter still. The tails produced from each round of amplification will be shorter and shorter until they are about the length of the primer. You will always have a background of longer tails, but as amplification proceeds each round will enrich the short-tail population relative to the long-tail population. A few more rounds and the short-tail population completely overwhelms the long-tail population and when the product is loaded on a gel you see one bright band (essentially the product from primer-length poly-A) and possibly a faint smear toward longer products.
OK, from a more experienced PCR person please -- is that on-track?
__________________ Jon D. Moulton, Ph.D.
Gene Tools, LLC [Only registered users see links. ]
Last edited by Jon Moulton; 05-21-2009 at 02:47 PM.