Temp. Gradient Gel Electrophoresis/GC clamps

molpath at chmeds.ac.nz molpath at chmeds.ac.nz
Thu Mar 23 03:40:01 EST 1995


In article <msnyder.57.2F6DEC34 at ace.acadiau.ca>, msnyder at ace.acadiau.ca (MARLENE SNYDER) writes:
> I am trying to figure out just how TGGE works, and why the GC clamp 
> facilitates TGGE detection of single base substitutions within a fragment.  It 
> seems to me the clamp would prevent melting, but melting is what is desired, 
> isn't it?  What am I missing here...
> 
> Thanks, 
> Marty Snyder

Yeah, melting is required, but only partial melting. DNA melts in domains, and
you want your mutation to be in an early melting part of the molecule. The 
easiest way to ensure this is to put a wacking great hunk of GCs at on end.
This bit will never melt, or at least will always melt last. What happens when
your molecule starts to melt, is that it forms a Y shaped structure that 
migrates only slowly in the gel. This molecule essentially stops moving and its
position in the gel depends on the temperature (or denaturant conc with DGGE)
at which the low melting domain melts. And this depends on its sequence/GC
content.
-- 
Andrew Fellowes                                             
Hospital Scientist                                          
Canterbury Health Laboratories, PO Box 151, Christchurch, New Zealand
 Ph: 64 3 364 0550 | Fax: 64 3 364 0525 | Email: molpath at chmeds.ac.nz



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