Bad primer design ? How can I know ? - How could I know if a primer would recognize a particular sequence ? (Sep/24/2007 )
Hi everyone ! 
I used the "search" function and read the "primer design tools" topic but I couldn't find the answer so...here I am. 
I have a problem. I have a single stranded DNA molecule and I use a pair of primers to amplify a particular region of this sequence.
These primers were designed by myself with online tools and I have to admit that I'm really not an expert in primer design.
Well, (remember that my DNA molecule is single stranded) I'm currently working on a rolling-circle amplification experiment (RCA).
quoting Kuhn et al. (Heiko Kuhn , Vadim V. Demidov , and Maxim D. Frank-Kamenetskii Rolling-circle amplification under topological constraints Nucl. Acids Res. 30: 574-580.) :
In RCA reactions, small single-stranded DNA (ssDNA) circles serve as templates for DNA polymerases (or sometimes RNA polymerases) generating numerous concatemerized copies of the circle. When a single primer complementary to the circle is employed, the accumulation of product proceeds in most cases linearly over time. An exponential (geometric) amplification via a so-called hyperbranched RCA (HRCA), also known as ramification or cascade RCA, is achieved by using a second primer with a sequence identical to a part of the DNA circle.
end of quote.
Since my forward primer sequence is complementary to the DNA template, it can anneal the DNA template and initiate the RCA process. But the other primer, the reverse primer is designed to be complementary of the RCA product (wich means that its sequence is identical to a part of the DNA template), so it's not supposed to anneal the DNA template and initiate the RCA process. The reaction is isothermal @ 30°C.
But I have a RCA product (observed Vs control made with no DNA template, no amplification) either with forward or reverse primer, wich is not supposed to be. 
Does anyone have an idea about this ? Could my reverse primer bind to another part of the DNA template, therefore acting as a forward primer ?
How could I analyze the affinity of my primers for my DNA template sequence (online tools ??) ??? 
If can know that my primer design is bad I can design new ones...but how can I know ?
Thank to anyone who will answer me. 
How could I analyze the affinity of my primers for my DNA template sequence (online tools ??) ???
If can know that my primer design is bad I can design new ones...but how can I know ?
Thank to anyone who will answer me.
More detail please. How big is your template? I presume you are confident of the sequence. How long are the primers? What annealing temperature are you using?
As to the reverse primer binding elsewhere, do you think it can bind anywhere else?
Can you try this: mix your template with the forward primer, dNTP and Taq and do 5 cycles of PCR-like reactions. Take 1ul of this and add to a reaction mix with both primers and do a touchdown PCR.
With isothermal reactions at 30 degrees, I'm not too surprised that you would get RCA initiation with the reverse primer. At that temperature, you may get annealling even though there may be mismatches between the primer sequence and the circle. If you have the sequence of the entire circle, I suppose you could try aligning this sequence with that of your reverse primer and see what you get. You could try relaxing the stringency of the alignment process and see how many more alignments pop up, if any.