DNA and autoclaving

Carl Simard c.simard at sympatico.ca
Thu Oct 22 17:09:55 EST 1998

theodorn at medlib.georgetown.edu wrote:

> Well, if I think way back to my school days, I remember learning about using
> Cot curves to examine the complexity of eukaryotic genomes, so therefore it
> _is_ possible to renature genomic DNA under the appropriate conditions. (Do
> they still teach this stuff, I wonder?  Maybe Patrick the grad. student will
> let us know)

You're right about Cot curves. But Cot curves are not experimental curves. They are
therotical curves based on the hypothesis that the DNA reassociation process is a 2nd
order kinetic reaction. So, you can apply 2nd order kinetics law to calculate relative
rate of DNA renaturation (you can check in GeneV, p660 in chapter 22).

    However, in real life you cannot see perfect renaturation of genomic DNA unless
you're very lucky. Just take, for example, a repeated sequence that is found in 100
copies troughout the genome. Once the DNA is denatured, each copy can reanneal with any
of the other one. The first copy will have 1/100 to anneal to his exact counterpart, the
second one will have 1/99, etc... For the correct (by correct, I mean "as in native
DNA") annealing of the 100 copies, you will have only 1/100! chances (it's 1/9x10E157).
You have to be very lucky to see your genomic DNA correctly reanneal!!! And repeated
sequences are found by thousand in the genome and they can each be found at hundreds to
thousands of copies!!! You certainly will have some renaturation of DNA after
autoclaving but you will never retrieved your native DNA.

> Also, I've never tried this, but the Trizol protocol describes restriction
> enzyme cutting the DNA isolated after Trizol treatment; surely this must be a
> case of renaturing genomic DNA after denaturation?

It's true that you can digest genomic DNA after Trizol but you don't denature the
genomic DNA during the procedure. You don't boil it and the NaOH 8mM you use at the end
is not alcaline enough to cause denaturation of DNA.

Carl Simard
c.simard at sympatico.ca

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