Quick Question About PCR ( I know, you're favorite topic )

[def jeff]

Ok, quick question about PCR. In the step where the DNA is heated to say 90 degrees C and separated, is this considered actually "denaturing" the DNA or something less than denaturing?

Totally just a question on terminology.

Thanks!

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[SomeDoc]

Absolutely. When the Rxn is cooled down, the newly separated complementary strands do not anneal back to their original confirmation; you may end up with hairpin loops, and various combinations of complementary annealings between one strand and itself, and with the original complementary strand.

 

[def jeff]

Really? That's what I began to think more about it. I definitely got hung up on the terminology of "denature" while working on my exam question, but you make perfect sense.

My test rationale was that it wasn't denatured because it could re anneal once cooled, and that to denature implied permanent loss of functionality. But I don't even think that's the case even with denaturing proteins ... can't proteins properly re fold once they have been denatured, albeit only under the correct folding conditions?

Sorry for the questions, I'm going to go through old books and google searches to try to figure this stuff out.

 

[SomeDoc]

Really? That's what I began to think more about it. I definitely got hung up on the terminology of "denature" while working on my exam question, but you make perfect sense.

My test rationale was that it wasn't denatured because it could re anneal once cooled, and that to denature implied permanent loss of functionality. But I don't even think that's the case even with denaturing proteins ... can't proteins properly re fold once they have been denatured, albeit only under the correct folding conditions?

Sorry for the questions, I'm going to go through old books and google searches to try to figure this stuff out.

Proteins can refold post-denaturation, but nothing like what the original configuration was originally. There are too many electrostatic sites in which the protein can fold into configurations other than the original. Protein folding occurs as a sequence of the function of DNA code; newly exposed electrostatic areas influence the folding pattern of newly synthesized a.a.'s, leading to the ultimate form. Refolding after denaturation can to haphazard, and multiple protein configurations.

 

[Monica Lewinsky]

Its not a terribly important point, but it might help you have a better idea of what is actually going on...

DNA unlike proteins will "melt" from the double stranded conformation to single stranded units when heated to ~90 degrees. It melts because hydrogen bonds are not strong enough to hold the strands together when the thermal energy of the DNA is that great. Analogously, proteins will denature at these high temperatures (usually much lower temps like ~45 as well though) and the tertiary and quaternary structure is lost. Proteins unlike DNA are synthesized in the context of ribosomes, ER, Golgi, and chaperone proteins (like hsp's) which help to form its higher order structure. Therefore when you cool a protein down from a high temp its not likely to regain its original conformation. DNA on the other hand is likely to anneal with complementary strands when cooled and this usually brings about the 'normal' dsDNA product (although hairpins can form, primer dimers in PCR, binding to other complementary sites, etc)