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Imagine a multiplex PCR in which after the extension step, the dsDNA is nicked and requires a ligation reaction to repair the nick before the next denaturation step. So the steps would be:

  1. Denature
  2. Anneal
  3. Extend
  4. Ligate
  5. steps 1-4 X 30

There are thermostable DNA ligase enzymes available (best activity at 65C), which don't get heat-inactivated by the high temp at the denaturation step.

It seems like this should work, but I can't find any info on anyone trying this. Any ideas?

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  • $\begingroup$ I don't get this. Why would you nick the dsDNA? $\endgroup$
    – Chris
    Jul 29, 2023 at 13:41
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    $\begingroup$ @Chris it's a multiplex PCR with 3 primers. Two flanking and one mutagenic in the middle. Assuming all 3 primers anneal, the polymerase will "bump into" the dsDNA in places where the other primers are hybridized (and either continue polymerizing or not depending on whether it's a non-displacing enzyme), but not covalently link the two ends. So before the next denaturation step those two pieces need to be ligated together. So the nick is an unfortunate byproduct of the PCR design that necessitates ligation. Now I'm doing in it separate rxns. I'd like to do it all in the same iterative reaction $\endgroup$
    – Ryan
    Jul 29, 2023 at 14:38
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    $\begingroup$ Ah, thanks. There are two problems I could imagine: First: The reaction may be hard to optimize in terms of the needed temperatures for annealing and the enzymes to avoid by-products. Second: Buffers needed for the different enzymes are incompatible. Or it is simply much easier to simply take the whole PCR reaction in the end, add some ligase and incubate it for an appropriate amount of time, so nobody ever bothered. $\endgroup$
    – Chris
    Jul 29, 2023 at 14:43
  • $\begingroup$ yes, the buffer compatibility was my main concern. My guess is that the ligation should be fine in the presence of pcr buffer, since as you mention, people just add dna ligase to their pcr rxns, post-pcr. But whether pcr is inhibited by ligation buffer I don't know. Looking at the buffer compositions it at least seems like it's worth a shot. Do you agree? Temperature I'm not so worried about, as there are ligases available with high optimum temps, i.e. right around my target annealing temp. They don't have inactivation temps, so my guess'd be that they won't mind high denaturation temp steps $\endgroup$
    – Ryan
    Jul 29, 2023 at 15:23
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    $\begingroup$ If the buffers are compatible (especially for the magnesium content), you could give it a shot. The Taq shouldn't have much activity at these "low" temperatures, so at least you shouldn't have to expect some unexpected here :-) $\endgroup$
    – Chris
    Jul 29, 2023 at 15:59

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In case this helps anyone, I found a technique called Gap Ligase Chain Reaction which includes both a thermostable ligase and a DNA polymerase enzyme in the same reaction:

Ligase Chain Reaction (LCR) --Overview and Applications (Wiedmann et al.) PCR Methods and Applications

Amplification of Chlamydia trachomatis DNA by Ligase Chain Reaction

Preliminary Evaluation of the Ligase Chain Reaction for Specific Detection of Neisseria gonorrhoeae

Detection of point mutations with a modified ligase chain reaction (Gap-LCR)

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