designing primers for Real Time - (May/11/2006 )
I´m starting my PhD and it´s about a viral disease in porcine. I´m designing a diagnostic test using Real Time based on SYBR GREEN. To do that I need to design primers and this is my big problem. I have tried all the free programs that are on the internet but none were good so I paid and now I´m using a good program that is giving me some little results because I don´t have primer dimers but what I have now are unespecific unions. I think this happens because this virus is really variable so, to amplify one of it´s ORFs I did a big alignment file on Clustal, and from it, I elaborated a consese sequence. Do you think is becasue of that?
If it´s because of the big alignment, do I need to make a smaller one?
I made a blast of my primers and they corespond to my virus (it´s the causative agent of Porcine Respiratory and Reproductive Sindrome)
What are those free programs that you used before? And what is the program you are using right now?
Is very important that your primer dont form primer dimer. However, certain degree of unspecific binding sometime can not be avoided (particularly to consenses sequence). How significant your unspecific binding is? what is the homology % (e value or score bit)?
If the unspecific binding is not significant, you might be able to eliminate it thgough optimization.