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.
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).