DNA Denaturation

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risingstar5
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DNA Denaturation

Post by risingstar5 » Fri Apr 21, 2006 8:06 pm

at what temperature does DNA denature?

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

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canalon
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Post by canalon » Fri Apr 21, 2006 9:21 pm

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
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Post by 2810712 » Sun Apr 23, 2006 8:23 am

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

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LilKim
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Post by LilKim » Sun Apr 23, 2006 7:08 pm

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)

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Post by 2810712 » Mon Apr 24, 2006 4:21 am

definitely, that's the ans.
THanks for quick reply.

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,

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Post by Jondice » Mon Apr 24, 2006 4:26 am

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

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Post by Jondice » Mon Apr 24, 2006 4:32 am

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

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LilKim
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Post by LilKim » Mon Apr 24, 2006 4:35 am

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.

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Post by Jondice » Mon Apr 24, 2006 4:38 am

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

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Post by 2810712 » Wed Apr 26, 2006 11:57 am

THanks for the link,

The positions,now i think, should ideally affect the rate of de/re naturation...is it?

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