I am collecting evidence, even anecdotal, how does single nucleotide deletion or insertion in primer region affect the outcome of real-time PCR. I am most interested in how much there is a delay in Cq (treshold cycle value) if compared to perfectly matched primer. But if you do not have Cq data, I also collect "Yes or No" answers, saying, whether there was amplification or no amplification.

I am interested specifically in insertion or deletion mismatches. (For substitution mismatches, there is a lot of data.)

I will appreciate any published or unpublished evidence.

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    $\begingroup$ where is the insertion from the 3'end ? Primer efficiency will be affected if point indels are introduced. If there is a mismatch at 3'end then extension may not happen at all.. As for data, nobody will deliberately design an RT primer with mismatches unless they are doing a research on primer efficiency.. :P $\endgroup$ – WYSIWYG Jan 22 '14 at 16:58
  • $\begingroup$ The insertion is at position 9 from the 3'end and the primer length is 19. The primer was designed to distinguish 2 related plant species, one was supposed to be amplified, whereas the other should not be because of insertion mismatch. I think that authors are wrong and that one deletion at such position is not enough to discriminate species. But I am asking people for empirical data, possibly something I can quote. $\endgroup$ – Barbara Jan 23 '14 at 8:51
  • $\begingroup$ This question is connected to my previous one (biology.stackexchange.com/questions/14357/…), but is a little different now. Before I asked about melting temperature, but now I know the Cq value delay is the parameter I am looking for (and is not correlated with Tm). $\endgroup$ – Barbara Jan 23 '14 at 8:51
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    $\begingroup$ you cannot distinguish between species accurately if your point mismatch is within the primer. Sequencing is the best thing to do but you should use a primer that binds upstream of variable region. Primer efficiency will change with mismatch which in turn will affect Ct-value, but there is no straightforward correlation between no of mismatch and Ct. $\endgroup$ – WYSIWYG Jan 24 '14 at 4:28
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    $\begingroup$ @ WYSIWYG Example of discriminating walnut from pecan by 4 mismatches in reverse primer. Recent study about various correlations between number of mismatches and delta Ct (and other correlations of other parameters). I do not expect similarly deep study about deletion mismatches, I am just collecting anecdotal evidence. $\endgroup$ – Barbara Jan 25 '14 at 10:00

As @WYSIWYG pointed out, having the indel difference in the 3' end can be informative, but I'm having a harder time finding evidence in the middle of the primer. Putting the mutation/indel at the 3' end will change the ΔCT. To do it well, you need wide coverage on your mutation site.

I think that it is possible that there could be a difference with a mismatch in the middle of a primer, but it wouldn't be as obvious as if it were closer to the 3' end. Unless there was some serious reason why you couldn't put it there (ie Tm below 45 on the reference) that sounds like poor primer design.

I will note I am certainly not a qPCR expert, and I don't think the question has been directly researched (as you surmised). Again when I've looked for Indels before (was doing some interesting stuff with HLA types...) we used larger primers for the qPCR.

If this doesn't answer your question satisfactory, is there an initial reference you would have us look at? I imagine things like good quenching and other factors could pull out some significance when comparing two templates (one with indel one without), but again, it's kind of odd to put the mutation in the middle.

Edit to comments: 1: I think the Cq delay is going to depend on things like length, sequence, and mutation. There may be a grand scheme formula/algorithm to predict the delay, but I don't know of one, and I couldn't find it if it does exist. Most of the graphs and differences I've seen have been big enough that it convinced me that it was significant. I don't know if this is like flow where you can sit and argue all day about the biological relevance of 40% to 30% of a particular group.

2: Sorry I didn't have time to come in and give an adequate summery of them. They are, as you pointed out, tangential to what you're trying to do, but do in fact address the problem through their methods. I will attempt a better summery as I have time (I've got a grant on I'm trying to get done by Tuesday, so probably after that). If you have any clarifications to the question to add, please do.

3: This may have grown from the general perception that it was needed, and that I aim for the highest Tm I can generally manage without structure problems being introduced. I also have the luxury of not needing to worry over the cost of ulta-mers. It also could have come from history of working in BACs years ago. Longer primers always increased my success rates when I was knocking out cassettes.

  • $\begingroup$ I just thught it must happen sometimes that the primer is designed, without knowing all possible variants of the target gene. And sometimes, I believe, it is accidentally discovered, that there is a single nucleotide deletion or insertion between primer and variant (allele) of template. I hoped somebody came across something like this during their work, and can tell me how much was Cq delayed. (Or, in classical PCR setup, whether there was amplification or not). The articles you pointed to me seem relevant, although not exactly answering my question. $\endgroup$ – Barbara Feb 1 '14 at 16:28
  • $\begingroup$ You know, I do not have time to read all the articles before my bounty expires. They do not seem to answer my question exactly, but they are relevant, and I was not able to find them myself before. And I do not want to waste the bounty... So let it be yours, but you might see me asking the same question different way later. Which I should do anyway, because most people seem to be confused by it. $\endgroup$ – Barbara Feb 1 '14 at 16:35
  • $\begingroup$ how did you know you need to use larger primers, when you are introducing indel-like point mutation ? Where does that original recommendation come from ? $\endgroup$ – Barbara Feb 1 '14 at 18:23
  • $\begingroup$ @Barbara sorry I just logged in and noticed your comments. I tried to at least address your comments some what. I will need to come back to address them further as I have time. $\endgroup$ – Atl LED Feb 2 '14 at 4:03
  • $\begingroup$ 1. " I don't know if this is like flow where you can sit and argue all day about the biological relevance of 40% to 30% of a particular group." - I did not understand this sentence. 2. "Sorry I didn't have time to come in and give an adequate summery of them" - I did not mean to say it was your responsibility to summarize (should one do it on this site ?), summarizing surely helps, but I am very thankful for raw citations. $\endgroup$ – Barbara Feb 2 '14 at 9:20

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