Primer-primer pairing: can I accept it? - (Aug/31/2005 )
I am working with Real Time RT PCR.
For the first time I need to design primers on my own.
I tested the primer (previously obtained with a software and checked on databases to be specific for my target) to check for inner pairing between forward and reverse primer at:
May I accept some internal pairing If they're not located at one end of the primer?
I think that if the pairing is between the 5' and 3' ends of the primer sequence it should not let polymerase to continue transcription.
Am i right?
I am afraid to have primer dimers if they have too many pairing between
Hoping I didn't make an obvious question
Thanks in advance
Are you using SYBR green for detection? SYBR green interacts with dsDNA in a non-specific manner, meaning that primer-dimers will cause your Ct to lower and thus make it appear as though you started with more template than was actually present.
Unfortunately primer-dimer prediction is a bit empirical. You should not have a problem if the pairing is between the 5' end and 3' end, but it practise it does not always turn out that way.
DNA sequencing software
The only way to know for sure is to buy the primers, use them in a conventional PCR and test for primer dimers by running the product on a 1.5% agarose gel. You'll probably get clean bands but if you don't, even after raising the annealing temperature, then you need new primers.
Thank you all for your answers, y re very kind.
yes, I am using Sybr green and I know it had this problem, Sp I did not want to use "dimering" primers!
I hoped some one had experienced some solution than try and see if it work
but seems it is still not clearly defined a safely way of design it until know!
I'll keep in mind your suggestions! thx a lot!