[Q] PCR gives extra band - heteroduplex formation?
bernard at elsie.nci.nih.gov
Wed Oct 18 23:25:56 EST 1995
First thanks to all who have replied to my original post.
The following thread has developed
In article <pnorton-1810951807080001 at norton1.jmc.tju.edu>,
pnorton at lac.jci.tju.edu says...
>In article <malexeyev-1610952020490001 at 184.108.40.206>,
>malexeyev at biost1.thi.tmc.edu (M. Alexeyev) wrote:
[Question basically asking if heteroduplexes should run at the average
of the two mobilities of the homodimers]
Just a reminder that my "mystery band" is running slower than either of
the two expected bands.
> I'm not quite sure that this can be predicted with certainty just from
>size. My experience running molecules that contain loops on _denaturing_
>acrylamide gels is that the the size of the loop matters somewhat, but
>that the porosity of the gel profoundly affects the mobility. I suspect
>that both parameters will affect the mobility of heteroduplexes and
>circular molcules on _native_ acrylamide, and possibly on agarose as well.
>(I don't think the original poster specified his gel system).
Yes, I did actually. 2% agarose x1 TBE. I don't do anything to the samples
just add the loading buffer and start it up.
> People have
>used the aberrant mobility of DNA molecules that contain bubbles or are Y
>shaped to localize origins of DNA replication by 2-D gels. Anyone out
>there using these techniques that can comment?
> If someone can offer a more precise, preferably more quantitative
>answer to the quoted post, I would like to hear it.
>Pamela A. Norton, Ph.D. Assistant Professor of Medicine
>Thomas Jefferson University
>Philadelphia, PA 19107 p_norton at lac.jci.tju.edu
I'd be interested as well. I am told that this "system" can be used to look
for mutations by hybridising wild type and potentially mutant PCR products.
Ambion have a kit (Mismatch Detect) that is based upon this (but you actually
look for RNA/RNA hybrids after in vitro transcription of the products).
I am going to follow a few of the suggestions;
a) Try and stop the reaction reaching a plateau (I'm currently at 35 cycles)
b) Try a polyacrylamide/urea gel for the products
c) Try and amplify the mystery band - should get all three bands as products
d) Cook up the two "pure" bands, anneal slowly and see if this reproduces
I'll let you know how it progresses. Thanks also to all who gave explanations
on how the heteroduplexes are/might be forming.
All the best,
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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