Longest Amplicon Using Real-Time PCR? - (Feb/05/2007 )
I am curious if anyone has attempted real-time PCR (Taqman) for amplification of a long PCR product (greater than 1kb)? I understand that the optimal amplicons are short in length. The reason I'm asking is that I'm amplifying genomic DNA with a specific forward primer and probe but a consensus reverse primer. The primers and probe were designed to amplify a short sequence but due to the consensus primer, there is the potential that an amplicon of about 1100bp could also be amplified.
I'd really appreicate if anyone knows of publications that address this, but replies of first-hand experience are also welcome!! Thanks.
*shiver* Brings back bad memories.
I thought about it, I tried it, I abandoned it. I tried for a large fragment (1.2 kb) and got virtually nothing. I reduced that to a 400 bp fragment and could detect my gene of interest as long as I had at least 2 million copies in the reaction! Even then the reaction was very inefficient as visualised by the graphs produced.
Bringing the test down to an approximately 100 bp product gives me excellent efficiency and I can detect between 10-100 copies in my sample.
Efficiency is a major factor in qPCR and generally speaking, long fragments amplify with a very poor efficiency for quantitative analysis.