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.
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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).
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.
