strand displacement activity assay

Aawara Chowdhury via methods%40net.bio.net (by aawara from pontiff-playground.org)
Wed Jul 8 09:33:59 EST 2009


In <714ed4af-7f75-4c0b-be18-c1c50dcdd963 from n11g2000yqb.googlegroups.com>,
 peter <peter.ianakiev 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
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