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closerehybridization of the plasmid is a simple explanation to the overestimation in quantitative PCR
Posted by jbonnet on 15 Apr 2010 at 13:13 GMT
If I have read correctly the paper, the authorsimply that supercoiling per se leads to an underestimation of plasmid copy number which translates itself in overestimation in PCR data if the supercoiled plasmid is taken as a standard.
The explanation is probably a bit simpler. Indeed, a supercoiled plasmid, cannot stay in a denaturated state, since the strands cannot separate. This leads to a strong inhibiton of the binding of the primers explaining the loss of efficiency in the first cycles of the PCR.
Obviously, this effect will not be seen on a linear plasmid or a plasmid relaxed by nicking. But the inhibitory effect should persist if the plasmid is relaxed by topoI since in that case, also, the strands cannot separate upon heat denaturation. such an experiment is reported in the paper by chen et al (ref 9).
So the oserved effect has probably much more to do with the covalent circular form than with supercoiling itself
However
A clear confirmation of this can be found
RE: rehybridization of the plasmid is a simple explanation to the overestimation in quantitative PCR
jbonnet replied to jbonnet on 15 Apr 2010 at 13:16 GMT
I unfortunately truncated my comment : an experimental confirmation of my statement can be found in the paper of chen et al cited as referenc 9