Hi Mark, iit seems that you are facing two different problems here if I understand well your post. 1. the PCR product is two large for a good quantification by QPCR and 2. I understand that there are several copies (4 copies?) of the same target in the genome, as it is in a gene cluster, which would make no sense for quantifying one gene product. Is it correct? If yes, try and design new primers following these guidelines: first align the DNA sequences of the different copies of the arc gene (use clustalW for instance), second find nucleotidic differences between copies such as one base is found only in the copy you want to amplify, third design one primer with its 3' last base lying on this base, fourth design the other PCR primer on the other strand about 80pb-150pb away the first primer (use Primer3 for designing a primer compatible with the first one).

Hi,

looking quickly to what is known about arcRACB cluster of H salinarum, it looks like my answer could have been much more simple. These genes are not copies of eachother. There are therefore plenty of nucleotidic divergence for designing arcA specific primers. Just design a short enough amplicon for a good QPCR result: say 150 bp is a max.

Thank you so much Jerome for your answer. Sorry for the misleading question though. That was very helpful. cheers.

Agree with Jerome. And for specific amplification, I normally design one of the primer pair based on the 3 prime or 5 prime UTR of your gene,