Bad primer design ? How can I know ?

[surt]

Hi everyone !

I used the "search" function 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.

[danfive]

If I understand you correctly this isothermal reaction takes place at 30C. That means that your R primer is most likely nonspecifically binding to the circular DNA. It will very likely be a waste of time to try to design a R-primer that won't nonspecifically bind at such low temperature.

[skatmbforum]

yea thats right 30c is vry low temp.for annealing & it will amplify nonspecifically

[wilson_trace]

Originally Posted by surt

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.

Hi,
I recommend using NetPrimer from PREMIER Biosoft: [Only registered users see links. ]
I have been using it for quite some time with reliable results. The program checks the primers for Tm, secondary structures such as dimers, repeats and runs.

Cheers

[mdjhomes]

Great content and very helpful thank and keep up the good work.