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Hello there!

After reading different sources regarding designing of qPCR primers, I'm a little confused regarding the concept of GC clamp.

Can you help me by telling which of these cases below is considered a GC clamp:

A) Any single G or C found at the last 5 nucleotides of the 3' end of a primer.

or

B) Any single G or C found at the very last nucleotide of the 3' end of a primer.

or

C) A couple of G or C (GG, GC, CG, or CC) found at the last 5 nucleotides of the 3' end of a primer.

Thank you!

Edit 1 in response to (Maximilian Press)

  1. Here in this book titled "Real Time PCR" Page 131 , based on table 7.1 one would say that option C (couple of G or C) is the correct one https://www.google.com.eg/books/edition/Real_time_PCR/-v-U-mXWg-gC?hl=en&gbpv=1&dq=gc+clamp+qpcr+primer+design&pg=PA131&printsec=frontcover

Table 7.1 Design criteria for real-time PCR primers, SYBR® Green detection

  1. And in the source by Steven Bradburn, PhD. which (Maximilian Press) has kindly provided the author explains that any single G or C base is considered a GC clamp regardless of its position as long as it is in the last 5 nucleotides of the 3' end which makes option A the correct one

https://toptipbio.com/gc-clamp-pcr/

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  1. Yet again in this conversation the contributor (Somanna Ajjamada) said: "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 6 bp at 3' end of primer"

Making a (couple of G or C) is considered a clamp and also a single G or C base is a clamp but only if the said single base is at the end of the 3' end and he also states that you should look at the last 6 nucleotides not 5 nucleotides as suggested by Steven Bradburn, PhD This makes option B correct

https://biology.stackexchange.com/a/80622/76328

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  • $\begingroup$ Can you clarify which sources you are looking at? It helps us to evaluate. For example this resource explains the idea and gives several examples. See also past questions on the topic: biology.stackexchange.com/questions/10843/… $\endgroup$ Jul 22, 2023 at 16:17
  • $\begingroup$ Thank you for response I tried to add resources showing discrepancy based on what I read elsewhere and what you have kindly provided in your response above. Waiting your opinion! $\endgroup$
    – H001O
    Jul 22, 2023 at 17:42

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I have never heard it being called a "GC clamp" but designing primers with GC near the 3' end is a standard way to make the 3' end duplex more stable, allowing for greater efficiency but possibly lower specificity. It is NOT a rule, just something to keep in mind. It seems some people interpret "near the 3' end" as 5 nt and others as 6 nt. I does not matter. What matters is Tm, possible secondary structures, primer dimers, too high AT content near the 3' end lowering the efficiency, too high CG content near the 3' end resulting in non–specific annealing, etc.

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