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I'm designing a set of primers and reading about the principles of primer design one of which is:

GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers (GC clamp) helps promote specific binding at the 3' end due to the stronger bonding of G and C bases. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.

From here.

My question is how essential is it to have a GC clamp?

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  • $\begingroup$ If it is important to have G or C why more than 3 G or C should be avoided ? $\endgroup$ – biogirl Oct 13 '13 at 15:05
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    $\begingroup$ @biogirl more G/C should be avoided because in an event of pairing between the primers you don't want the pairing to be too strong. Otherwise you'll get a lot of primer dimers. Ben, GC clamping is important as it will allow strong bonding at the 3' which is good for elongation but this is not the primary parameter to be tweaked. There are other important parameters to handle (for e.g self complementarity, low-complexity etc). $\endgroup$ – WYSIWYG Oct 13 '13 at 16:12
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It's hard to provide an objective answer. If you have a decent length and good complexity, even a single terminal 3' G or C would do. Of course, one has to take into account the primer's overall GC:AT ratio and things like annealing temperature.

Here's a link to diverse opinions on the topic and it has this nugget (which I subscribe to when possible):

FWIW, my preferred offerings to the PCR gods are primers with a single G or C 3', FWIW. Seems to keep 'em happy most phases of the moon.

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I will offer my own (empirical) account of primer design. A GC-clamp aids in specificity of the priming and therefore contributes to the overall efficiency of the PCR reaction. In the past, I have (out of necessity) designed primers that had both too much GC-clamping on the 3' end, and used primers without any GC-clamping. These PCR reactions were performed successfully, with differing levels of primer-dimer formation and overall efficiency.

In my experience, GC-clamping is nice, but not strictly required for good PCR. My general rule-of-thumb is to terminate my primers with 2 G/C wherever possible. If something is going to fail, it is not usually the PCR.

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GC clamping at 3', that is having a single G or C at the 3' end or a couple of G/C within the last 6bp at 3' end of primer, can help retain the primer on the template during elongation, because of stronger bonding compared to A=T. This is not mandatory; it's one of the characters of the primer which might improve your PCR reaction.

At the same time, avoid a sequential combination of GC in the last 6bp which might lead to self-dimers.

Example:
5'-.....GCGC-3' has higher chance of dimer formation than 5'-.........GCCG-3'; in such cases you can still use the latter primer sequence.

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Unless you are PCRing something you know to be challenging, just use primer3 and don't worry about it.

http://bioinfo.ut.ee/primer3-0.4.0/

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