competition of multiple PCR products - (Aug/15/2006 )
I have a question regarding the competition of PCR products. I'm looking at a hypothetical alternatively-spliced gene product that is susepcted to be produced at roughly 20-fold lower than the primary full-length transcript. I have designed a PCR hoping to pick up both products, but don't pick up the alternatively-spliced product. My dillemma is that I can not be sure whether this is because it is not there (I can't design specific primers as at the moment - we're hypothesising about it, we can't actually prove it exists), or whether the primary transcript "competes" the alternatively spliced transcript.
So does anyone know what sort of ratio you can have before you would mask a minor variant by competition? Does 1 in 20 sound feasible? The product sizes are hypothesised to be 2.5kb and 2kb.
sorry for posting twice - my browser crashed the first time!