Primer-primer pairing: can I accept it? - (Aug/31/2005 )

Hi all,

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:

http://www.operon.com/oligos/toolkit.php

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
theirselves.

Hoping I didn't make an obvious question

Thanks in advance

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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.

-Hank

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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.

Daniel

DNA sequencing software

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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.

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Thank you all for your answers, y re very kind.

Anyway:

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!

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