strand displacement activity assay

peter via methods%40net.bio.net (by peter.ianakiev from gmail.com)
Wed Jul 8 11:58:45 EST 2009


On Jul 8, 10:33 am, Aawara Chowdhury <aaw... from pontiff-playground.org>
wrote:
> In <714ed4af-7f75-4c0b-be18-c1c50dcdd... from n11g2000yqb.googlegroups.com>,
>
>  peter <peter.ianak... from gmail.com> wrote:
> > Hello fellow scientists,
> > I am trying to come up with an assay to determine strand displacement
> > activity of T4 DNA pol. Any ideas? In general it is accepted that T4
> > pol has very little strand displacement, but for my application I need
> > complete lack of such (single molecule assay). Thanks in advance old
> > timers....
>
> I've done such assays; I don't know if the sensitivity is suitable for
> your needs, but this is one way to get it done:
>
> a) Make a ssDNA closed circle which will be used as template.  It is
>    important that this be a closed circle, so that the 3'->5' exo
>    of T4 doesn't chew it up.  These days, I imagine you can make a
>    long single-stranded oligo that has a modified 3'end, so that
>    exonucleolytic degradation cannot occur. Use this long oligo
>    as template.
>
> b) Anneal two oligos to it - one of which is radioactively labeled,
>    and ends in a 3' phosphate, not 3' hydroxyl.
>    This oligo must be about approximately 10 nucleotides 3' to then
>    3' end of the other oligo.
>
> c) Add T4 DNA pol and dNTPs.  Electrophorese the reaction by
>    non-denaturing PAGE of suitable percentage.
>
> d) The annealed radiolabeled oligo will run near the top of such
>    a gel.  Any displaced radiolabeled oligo will migrate faster.
>
> Good luck,
> A
> --
> Email: echo 36434455860060025978157675027927670979097959886449930P | dc

Thanks for the suggestion,
Unfortunately I don't have permit for radioactivity in this lab....I
was thinking something along annealing oligo that have a point
mutation that would abolish restriction site and then making dsDNA
with another oligo . If it gets displaced it will be cut with the RE,
if it does not get cut - oligo was not displaced. That is assuming
100% annealing though ....


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