DNA structure and PFGE

Phillip San Miguel pmiguel at bilbo.bio.purdue.edu
Tue Jan 20 10:46:18 EST 1998


I think this is wrong -- supercoiled plasmids will be more shear-resistant
than linear DNA. Small plasmids aren't much effected by pulsed field so they
migrate they same way they would on a normal gel.  So they end up looking
enormous compared to linear DNA.  I remember a paper by Bruce Birren (I think)
about using pulse-field gels to size large supercoiled plasmids (probably
BACs).  I can't find the reference in Medline though... But here is a
reference for supercoiled DNA in pulsed-field gels:

Authors
     Mathew MK. Hui CF. Smith CL. Cantor CR .
Institution
     Department of Genetics and Development, College of Physicians and
Surgeons,
     Columbia University, New York, New York 10032.
Title
     High-resolution separation and accurate size determination in
pulsed-field gel electrophoresis of DNA. 4. Influence of DNA topology.
Source
     Biochemistry. 27(26):9222-6, 1988 Dec 27.
Abstract
 Pulsed-field gel electrophoresis is a powerful technique for the
fractionation of linear DNA molecules with sizes above 50 kilobase pairs (kb).
Here it is demonstrated that this technique is also effective for separating
smaller DNAs including linear, circular, and supercoiled species. The
mobilities of linear DNAs larger than 8 kb can be modulated by pulse times
between 0.1 and 100 s. The mobility of supercoiled DNA molecules up to 16 kb
is generally unaffected by these pulse times except that 10-s pulse times
cause a small but distinct increase in the mobility. The general insensitivity
of small supercoiled DNAs to pulse time presumably occurs because these
species reorient so rapidly that they spend most of their time undergoing
conventional electrophoresis. However, the mobilities of larger supercoiled
DNAs are affected by pulse times of less than 1 s, and at 0.1 s the molecules
are better resolved by pulsed electrophoresis than by ordinary
electrophoresis. The mobility of 3-19 kb nicked and relaxed circular DNA
molecules is also affected by pulse time but in a complex way.


Edgar Valencia wrote:

> Hi netters:
>
> I know PFGE can be use to se large linear molecules but not big plasmids
> and I guess this is due to the fact than a circular molecule will be
> broken during the migration inversions.
>
> OTOH I commonly use the Eckhardt technique to see large circular plasmids
> (150-1200 kb in size). I've been told that this technique IS NOT able to
> show linear molecules (but Im not sure if this is true).
> My questions are:
>
> Which is the maximum size of a circular molecule which could survive in a
> PFGE?
> What would happen to a circular molecule in the gel? Am I going to see the
> fragments as a smear all over the lane or the circles won't enter the gel?
>
> Could I use PFGE/Eckhardt's technique to distinguish among linear vs
> circular molecules or depending on the size I'll see both in the gels?
>
> Is there any other technique which could help me to distinguish among
> linear/circular HUGE plasmids which doesn't be electron microscopy?
> Perhaps a nuclease+electrophoresis assay?
>
> Any help/references/comments will be appreciated.
> TIA
>
>      ###################################################################
>      #                                             ______              #
>      #                                             \     \____         #
>      #       Edgar Valencia-Morales        ********]|-----|___\____    #
>      #  Departamento de Genetica Molecular ********]|_______>______/   #
>      #           CIFN - UNAM                        /______ /          #
>      #             MEXICO                                              #
>      #     e-mail edgar at cifn.unam.mx                                   #
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