## DNA Denaturation

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

risingstar5
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### DNA Denaturation

at what temperature does DNA denature?

which base pairs have a higher denaturation point? - guanine/cytosine or adenine/thymine?

canalon
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G/C are more stable than A/T pairs. The denaturation point depends on the whole sequence. But when you want to get sure to denature the molecule, like in PCR, you just go to 94C
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

2810712
King Cobra
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HOw much is the temperature of complete separation of two strands different for diff. sequences...or how the fraction separated in given time vary with temp..? IS the variation so obvious?
help Conlo, help...

hrushikesh

LilKim
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think about the structure of A-T base pairs and C-G base pairs.

there are 2 bonds that form between each AT pair and there are 3 bonds that form between each CG pair. Therefore it will require more energy (heat) to break CG versus AT.

So, sequences containg more G's & C's will require more "Heat" to be "melter" in comparison to sequences that are mostly A's & T's.

If you're comparing the sequences of two oligo. you can estimate the "melting temp."
- For every C/G you add 4 degrees C
- For every A/T you add 2 degrees C

For example You have a primer sequence "CTTAGC ..."
you'd add: 4+2+2+2+4+4 (+"...")= 18 degrees C (+"...")

(2810712 - i'm not sure if this is the answer that you're lookng for)

2810712
King Cobra
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definitely, that's the ans.

I think , the positions of the A/T & G/C along the double strands will also matter a bit...but not that much difference for given organism for sufficiently long DNAs, right?

thanks,

Jondice
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Surely LilKim's equation for estimating a strand's denaturation temp isn't linear indefinitely; I must have missed something somewhere =p.

Jondice
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I found this from doing a quick google search, I think you'll find it quite helpful:

http://www.cbs.dtu.dk/staff/dave/genomi ... nature.pdf

LilKim
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As far as the denaturation.. as Canalon said a standard temp. for denaturing genomic DNA is 94 degrees C.

However, if you're using an oligo (approx. 20 nt long) you can used that Melting temp. equation to estimate. (it's worked well for me .. for PCR stuff ....Using approx. melting temp is essential for maintaining stringent PCR condtions and preventing non-specific amplification)

As far as postion of a/t and g/c ... I don't know if there is a difference for melting temp relative to postion.

... this is probably waay to much info.

Jondice
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ah, yes, you said oligos, my bad there.

2810712
King Cobra
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