How is primer annealing, and, consequently, PCR amplification affected by single nucleotide deletion or insertion inside the primer ?

Imagine a primer like this:
and the corresponding part of template DNA has one G missing, so it looks like this:

The possible pairing could be
or anything in between.

Is it possible that amplification with such primer would be completely disrupted in normal real time PCR at 60°C annealing? Could this completely disrupt the amplification?

If the mismatch was substitution-like, I would be pretty confident, the primer would be still functional and amplification would occur. In the extreme case, it would be at least residual amplification at late Ct. There are a lot of data, how substitution mismatches affect primers and I also have a lot of personal experience with that.

Unfortunately, the deletion mismatches are less studied plus googleproof. The only indication I have found is this work:
Lipsky RH, Mazzanti CM, Rudolph JG, Xu K, Vyas G, Bozak D, et al. DNA melting analysis for detection of single nucleotide polymorphisms. Clin Chem. 2001;47:635-44.
In that work, single nucleotide deletion had similar or lower affect on melting temperature than substitution mismatch. But it was about longer oligos. Examples:
Effect of deletion:
133 bp fragment, 67 % GC, deletion SNP at position 43, delta Tm (homo-hetero duplex) = 1.2°C
Effects of substitutions:
152 bp fragment, 43 % GC, substitution T to C at position 68, delta Tm (homo-hetero duplex) = 0.9°C
100 bp fragment, 41 % GC, substitution T to C at position 42, delta Tm (homo-hetero duplex) = 1.4°C
163 bp fragment, 60 % GC, substitution C to T at position 86, delta Tm (homo-hetero duplex) = 2.2°C
110 bp fragment, 59 % GC, substitution G to A at position 66, delta Tm (homo-hetero duplex) = 3.8°C

1. Can you recommend me literature about how deletion mismatches inside (not at the very end of !!!) primers affect annealing and PCR ?
2. Would You guess the primer in my example would be still functional , at least partly, or would You expect no amplification at all ?

EDIT after your inputs:
This online application " mfold.rna.albany.edu/?q=DINAMelt/Two-state-melting " thinks, deletion mismatches are more destabilising than substitutions, at least for short primers. For my own example, it calculated delta Tm (homo-hetero duplex) = 12.9°C . If I try substitution mismatches instead, delta Tm (homo-hetero duplex) is in interval 3,8°C to 5,7°C.

New question
If you have experience as similar as possible to my case, which is 19 nt long primer with single nucleotide deletion in the comlementary template at position cca 6 - 9 from 3' primer end, annealing temperature used at 60°C, please let me know if You achieved amplification or not. Please, give me a respective reference, if you have it, so I will quote it in my review :-) .

Also, I am still interested in general info, as long as the topic is narrow enough to be about single nucleotide deletions or insertions in primers. (Not substitution mismatches).

  • 2
    $\begingroup$ primers would be functional as long as the 3'ends match properly and the mismatch is not huge. You can calculate the changed melting temperature using UNAfold DNA-DNA hybridization server. $\endgroup$
    Jan 9, 2014 at 16:54
  • 1
    $\begingroup$ Very neat application, thanks ! For the benefit of others - I have found where exactly one enters the sequences at the server: mfold.rna.albany.edu/?q=DINAMelt/Two-state-melting $\endgroup$
    – Barbara
    Jan 10, 2014 at 8:49
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    $\begingroup$ Similar to @Chris, I've used longer, even ultra-mers to cause deletions. I've been curious about how small I could make the primers, but I've always had the luxury of being able to make them pretty long without worrisome 2ndary structures. Have you considered trying this on a test template to see how it goes? Wouldn't be too costly to test (I've considered it). $\endgroup$
    – Atl LED
    Jan 28, 2014 at 2:28

2 Answers 2


We used this kind of primers to generate out of frame mutations or to add additional bases. In my experience your PCR will work (probably a a lower efficiency) and you will get a product with an additional base. We used primers with bigger differences in PCR based site directed mutagenesis of plasmids, there up to 10 bases didn't match but the primers where also longer. For single nucleotide mismatches (either + or - one base) we used primers around this size.

Regarding the literature, this publications might be useful:

Especially the first publication contains a lot of other interestings references.

  • 1
    $\begingroup$ If you do not wory about your identity, would you mind sending me a reference for your publication, where there was primer of this size (cca 19 nt), had a single nucleotide deletion or insertion, and still worked ? ( Iknow I am probably too demanding, because you have sent me 4 publications already. It would just be convenient to have reference for this specific example right away). $\endgroup$
    – Barbara
    Jan 9, 2014 at 13:44
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    $\begingroup$ No big deal: Look at this paper, the sequences are in the supplemental data. The shortest primer pairs are 25mers. The length is dictated by the surrounding sequence. $\endgroup$
    – Chris
    Jan 9, 2014 at 15:03
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    $\begingroup$ @Barbara What I forgot: I recently used sligtly shorter primers (21 and 22mers) but this results are not published yet. $\endgroup$
    – Chris
    Jan 9, 2014 at 16:02
  • $\begingroup$ @ Chris: Thanks, at least I have assurance, it can work this way. But if by chance you'll remember you have a poster presentation where those short primers are mentioned, please, let me know. $\endgroup$
    – Barbara
    Jan 10, 2014 at 9:02
  • $\begingroup$ @ Chris again: The articles you listed say nothing about single nucleotide deletions, with the exception of Kwok 2014, which says minimum. (Kwok says, the primers can be used to indtroduce indel SNP and that they should be placed in the middle of 24 - 36 base oligo.) Your own article mentions only long (46 nt) oligos spanning 3nt indel. Am I overlooking something ? $\endgroup$
    – Barbara
    Jan 10, 2014 at 16:54

Adding another reference. This group looks at the effect of different mismatches (e.g. A>T vs A>G) and also looks at positional effects.

Our results show that single mismatches instigate a broad variety of effects, ranging from minor (<1.5 cycle threshold, eg, A–C, C–A, T–G, G–T) to severe impact (>7.0 cycle threshold, eg, A–A, G–A, A–G, C–C) on PCR amplification. A clear relationship between specific mismatch types, position, and impact was found.


  • $\begingroup$ I was interested in deletions, not substitutions. No matter how much stress this out, people keep sending me info about substitutions in every forum ever. $\endgroup$
    – Barbara
    Mar 8, 2018 at 3:43

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