I have a mixture plasmids and undesired short linear fragments that share the same sequences. During denaturation and annealing, I would like the plasmids to 'find each other' before annealing to the shorter linear fragments. Assuming the concentration of shorter fragments is significant, is there a temperature profile to bias towards re-annealing of longer DNA?

More specifically, this is for a variation of the Surveyor Mutation detection assay, where re-annealed DNA with mismatches are digested, leaving non-mutant DNA intact. I would like to keep non-mutant plasmids for E. coli transformation. However, some linear fragments ~10-50% of the length of the plasmid have escaped exonuclease treatment and would compete with the plasmids during annealing.

  • 2
    $\begingroup$ it is unclear what you ask. And why not transform e. coli with your mixture, only circular plasmids will propagate and linear pieces will be degraded/purified-out $\endgroup$ – aaaaaa Apr 21 '15 at 6:51
  • $\begingroup$ If the linear fragments anneal to the circular plasmids instead of the complement, the resulting DNA would only be partially double-stranded, which I assume would render it unusable by bacteria. This would lower the transformation efficiency. $\endgroup$ – bravetang8 Apr 21 '15 at 20:20
  • $\begingroup$ did you think of just using DNAse that targets ssDNAs? For example, Mung bean nuclease. There should be another enzymes with similar activity, e.g. S1 $\endgroup$ – aaaaaa Apr 21 '15 at 22:07
  • $\begingroup$ I'm using PlasmidSafe which is a cocktail of exonucleases that do that. It's just not 100% efficient and I wanted to leave no room for error. $\endgroup$ – bravetang8 Apr 22 '15 at 0:30

Short answer is that higher temperature favors annealing of longer sequences. There are number of ways to calculate melting temperature, but all of them produce similar results: longer polymers require more thermal energy to melt. Hence, quick cooling from higher (say, from 95C thermocycler can cool in 10-12 sec) to RT/4C will favor re-annealing of circular strands. Slower cooling should allow more ssDNAs to bind.

But again, if end goal is to select for circular dsDNA, simple transformation should take care of that.

  • $\begingroup$ Thanks for the answer! How about slow annealing down to slightly below the melting temperature of the plasmid and then snap cooling on ice from there? $\endgroup$ – bravetang8 Apr 21 '15 at 20:34
  • $\begingroup$ Also, unlike oligonucleotides, longer DNA is flexible and contains local regions of higher and lower melting temperatures. Would the the overall melting temperature be accurate in this case in describing that half of the ssDNA molecules have completely annealed? In other words, would it be common to have two ssDNA molecules incompletely annealed to the same template, where one of the molecules is partially blocking the other one? $\endgroup$ – bravetang8 Apr 21 '15 at 20:47
  • $\begingroup$ I don't know, what is really possible. What is true, is that at any temperature interaction that includes more bases is more favorable. Think about it as a dynamic equilibrium: at each moment ssDNAs will bind/try to bind and unbind. However, it takes only dozens of bases to unbind whole oligo, whereas circular DNA is on order of KBs. So oligo will unbind and go into solution much easier than second strand of circular dsDNA $\endgroup$ – aaaaaa Apr 21 '15 at 22:03

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