What Is A GC Clamp In PCR Primers?

In this article, I will explain what a GC clamp is in relation to PCR primer design and why they may be useful to include in your primers.

More information regarding GC clamps and detailed qPCR primer design instructions can be found in our online course: Mastering qPCR.

What is a GC clamp in PCR primer design?

Simply, a GC clamp is the presence of a guanine (G) or cytosine (C) base in the last 5 bases (the 3′ end) of a PCR primer. Having the presence of a GC clamp in a PCR primer can help to improve the specificity of primer binding to the complementary sequence.

The figure below shows an example of a GC clamp in a PCR primer. Note, the GC clamp is highlighted in red (ie the C base).

GC clamp example

Other examples of a GC clamp (in red) in PCR primers include are listed below.

  • 5′-CTCTGTAGGGTCGCGACTAC-3′
  • 5′-CGCTACCACCATCGATTGAT-3′
  • 5′-GGATCTGGCTGCATGCTATG-3′

Notice that it does not matter where in the last 5 bases the G or C base is in order for them to be referred to as a GC clamp.

How a GC clamp can improve PCR primer specificity

Binding between G and C bases is formed by three hydrogen bonds, compared to only two between adenine (A) and thymine (T) base pairs. Therefore, G and C base pairs are considered to have stronger binding than A and T base pairs.

Since G and C base pairs have superior binding, placing 1-2 of these bases at the end of the primer will encourage complete primer binding. However, it is not recommended to include >2 G or C bases in the last 5 bases of a primer. Doing so can actually have adverse effects by increasing the primer melting temperature (Tm) and reducing primer specificity.

Conclusions

In sum, a GC clamp is often recommended during PCR primer design in order to encourage complete primer binding to the complementary template. However, too many G or C bases especially at the end of primers can have negative effects.

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