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Are there ways to make an oligo displacement-resistant?


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#1 MGDrzyzga@brandeis

MGDrzyzga@brandeis

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Posted 12 December 2011 - 02:45 PM

Short version: I'm looking to block an undesired step with a displacement-resistant oligonucleotide that is also incapable of acting as a primer during a polymerase step.

Long version: I've been struggling with a strand displacement step. I've got a DNA oligonucleotide containing an AT rich hairpin loop, folded over and annealed to itself in a GC rich region. My polymerase being used for strand displacement has been causing issues with not reliably displacing the hairpin on the first go, creating a contaminant where the enzyme first follows the hairpin around before doing the desired displacement. Due to follow-up work, even a small amount of this undesired contaminant compromises the goal of this setup. I've been exploring ways around this and come up with many dead ends. One idea was using a blocker of some sort that also binds in the hairpin, preventing the polymerase from following the entire hairpin around. My first idea is using a PNA oligo that binds at this second site in the hairpin. Question is, can I reasonably expect a short sequence of PNA to effectively block this route? Or is it enough that the PNA be harder to displace that a GC rich region, and thus point my polymerase in the right direction?

For extra help, imagine the DNA as a clock, with dsDNA coming off at 3 o'clock, and ssDNA going around the rest of the clock. I've got a primer that anneals around 4-6 o'clock, and I want it to go off to the side at 3 o'clock rather than continue back to 2 o'clock. I'm looking for ways to bind something at 2 o'clock to prevent the polymerase from going back that way.

FYI, I have looked at switching to other enzymes for the strand displacement. Unfortunately, I've yet to find something strong enough that doesn't cause subsequent mayhem by not being easily inactivated.

Thanks for any advice.




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