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I'm developing software which, among other things, designs primers pairs (forward and reverse) for qPCR. In my research on various properties, I read about GC-clamps and I understand the basics of it I think. There is still something unclear to me.

Do both primers in a pair need to have a GC-clamp in order for it to be effective? Or does one GC-clamp in, let's say the forward primer, just increases specificity, and both primers containing a clam increasing it even more?

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  • $\begingroup$ The new question is too Health'y for me to answer. Very nice edits for Health though. Likely others can answer it. $\endgroup$
    – AliceD
    Commented Apr 21, 2016 at 13:12
  • $\begingroup$ If we are adding gc clamp in both primers, the sequence should be different or same? $\endgroup$ Commented Nov 3, 2021 at 10:09

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You can have GC-clamps (3' terminal G/C) in both primers; this may overall improve the PCR efficiency. However you should take care not to put too many G/Cs. This may lead to stabilization of primer pairs, leading to formation of primer-dimers. Also see this post: When designing primers how important is the GC clamp?

GC-clamps are not always essential and you should look at other parameters too.

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  • $\begingroup$ So, in summary: A clamp in one primer is good for efficiency and clamps in both are better (with the right amount of G/C ofcourse). So I don't need a GC-clamp in both of them. Is this right? $\endgroup$
    – Iarwain
    Commented Mar 22, 2016 at 13:25
  • $\begingroup$ @TomKral Not strictly necessary but good if you have it in both. $\endgroup$
    – WYSIWYG
    Commented Mar 23, 2016 at 4:44

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