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Alkaline denaturation of prokaryotic DNA

Alkaline denaturation of prokaryotic DNA - Basic Lab Protocols and Techniques

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Old 08-04-2006, 06:09 AM
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Default Alkaline denaturation of prokaryotic DNA



Hi everyone, I'm a high-school student who needs help understanding some basic principles in the chemical properties of DNA.

One of the methods used to extract plasmids is alkaline denaturation of DNA followed by renaturation of plasmid. I've been told that because plasmids are circular and un-nicked, they renature and stay in solution while the larger chromosomal DNA, being linear, would anneal haphazardly, causing it to clump and precipitate out of solution.

But what I've read in textbooks is that chromosomal DNA of prokaryotic cells are circular too, not linear. So shouldn't the method above allow chromosomal DNA to re-anneal like plasmids, thus remaining in solution?

Thanks in advance!

Andrew
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Old 08-04-2006, 05:45 PM
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Talking Re: Alkaline denaturation of prokaryotic DNA

Hello!,

I would say that is a great question.

Alkaline lysis is the preferred method for the isolation of closed circular plasmid DNA from bacteria.

This method is based upon the fact that plasmid DNA, unlike the chromosomal DNA, is able to rapidly anneal following alkaline denaturation.

Also it is based on the fact that potassium dedecyl sulfate, compared to its sodium counterpart, has a much lower water solubility.


Basically, the detergent functions to lyse cells and denature proteins and the alkaline conditions denature chromosomal DNA, plasmid DNA, and proteins.

Upon neutralization and high salt conditions, only the supercoiled plasmid DNA renatures and remains in solution.
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Actually, your question is BEST answered by the following:

* 1) Chromosomal DNA strands can separate, whereas highly supercoiled plasmid DNA cannot.

* 2) Chromosomal DNA can be centrifuged away as it stuck to the cell membrane, plasmids are free.

Explained here:

Under highly alkaline conditions, both of the strands in non-supercoiled DNA (linear fragments of chromosomal DNA, relaxed and nicked circular DNA)
separate and become partially removed from solution. However, separation does not occur with supercoiled forms of plasmid DNA.
This is because the two strands are intertwined and entangled in such a way that they cannot come apart or relax.
Therefore, supercoiled plasmid remains free in solution.


Potassium acetate neutralization buffer contains acetic acid and
potassium salts. The acidic buffer neutralizes the alkaline conditions
created by the sodium hydroxide. The potassium causes the SDS, with
its associated membrane fragments and proteins, to precipitate. The
chromosomal DNA of E. coli is attached at several points to the cell
membrane. Centrifugation of the potassium-SDS-membrane complexes
also removes large amounts of entrapped chromosomal DNA.


Reference
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Hope that helps, I remember my Molecular Biology Labs now!
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Old 08-04-2006, 05:50 PM
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Default Re: Alkaline denaturation of prokaryotic DNA

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Another reference that was helpful for me also!
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alkaline , denaturation , dna , prokaryotic


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