2 questions based on DNA finger printing- TBE Buffer

[cruiseforkidman]

Hello Everyone,


First question:
please suggest an explanation for the presance of each component in TBE buffer- for the use of DNA fingerprinting


Second Question:
If DNA that has been run on agrose gel is denatured by removing the gel from a electrophoresis chamber and soaking the gel in denaturayion soution (1.5 M NaCl/0.5 M NaOH)

Firstly how would i prepare 400ml of this solution, given a bottle of solid NaCl and 5M solution of NaOH (MW of NaCl is 58.44).
please provide workings so i can understand clearly how this is to be done.

And secondly how does this solution actually acheive DNA denaturation?

Thanking each one of you.

[moleculardude]

Wow... this looks like a multi-question essay or something you get in school...

Hope you donate lots of points for this...

Ok here goes a few.

Denaturation of DNA


Alkaline lysis (and Alkaline Denaturation) is the preferred protocol isolating DNA and even RNA from cells.

It a very useful method to quickly, reliably and cleanly obtain DNA from cells.

NaOH denatures the DNA. Cellular DNA becomes linearized and the strands are separated.

NaCl or Salt

DNA is precipitated by salts such as NaCl (sodium chloride). This is because DNA is negatively charged, and the addition of salt masks the charges of DNA and prevents water interaction (phosphate backbone and water molecules - see water bridge for DNA) and allows DNA to denature - more salt and you get precipitation.



I guess you are doing a Southern Blot for DNA analysis.
in this case:

Soaking the gel in NaOH results in the two DNA strands separating. This allows the probe to hybridize to single stranded DNA.



I believe the salt breaks the interaction of DNA with the water molecules (by interacting with the phosphate backbone) and the NaOH denatures the DNA by separating the DNA strands (through disruption of the hydrogen bonds).

Now to explain more:


DNA is composed of two DNA strands and these strands are basically held together by hydrogen bonding interactions.

Hydrogen bonding interactions can be quite weak as heat, acid, and bases can all disrupt H-bonding and cause the DNA strands to separate.

As you did in your lab, NaOH is used to extract DNA from cells and to denature it (precipitate it as well).

The best way to understand what is happening at the atomic level is to see the molecular 3-D structure of DNA. What is missing also is the water molecules that create a bridge between the DNA hybrid in the groove and solvate it ( ie make it solube in water solution )


Some References

Charles A. Zittle. Enzymatic hydrolysis of deoxyribonucleic acid prepared with the use of strong sodium hydroxide. Journal of the Franklin Institute, Volume 245, Issue 1, January 1948, Pages 78-80.

John S. Ullman and Brian J. McCarthy. Alkali deamination of cytosine residues in DNA. Biochimica et Biophysica Acta (BBA) - Nucleic Acids and Protein Synthesis, Volume 294, Issue 3, 4 February 1973, Pages 396-404