DNA denaturation during PCR ??

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bvk_1983
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DNA denaturation during PCR ??

Post by bvk_1983 » Fri Jun 30, 2006 8:51 pm

HI

during PCR once the dsDNA template is denatured to ssDNA the temp is decreased to allow the primers to anneal, but why doesn't the ssDNA template re-anneal ?

Is it because of fast cooling .... or some other reason !!!

oppox
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Post by oppox » Fri Jun 30, 2006 10:11 pm

without primers they would but u got so much more primers then template so its just a matter of probability

Monday
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Post by Monday » Sat Jul 01, 2006 2:45 am

The concentration of primers is much more higher than that of temp DNA.

bvk_1983
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Post by bvk_1983 » Sat Jul 01, 2006 9:36 am

One may make nucleic acid (NA) single-stranded for the purpose of annealing - if it is not single-stranded already, like most RNA viruses - by heating it to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling it: this ensures the "denatured" or separated strands do not re-anneal.
(http://www.mcb.uct.ac.za/pcroptim.htm)


any comments on the above statement

oppox
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Post by oppox » Sat Jul 01, 2006 10:52 pm

well its a way, but an ordinary pcr machine cant flash cool the sample. If u mix DNA and primers heat it to 95 and the transfer it to dry ice or something u get the effect, but it takes quite some time if use a non robotic pcr machine to cool it to the annealing temp. Im not an expert on this but I thought that in an ordinary pcr reaction it depends on the primer conc.

bvk_1983
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Post by bvk_1983 » Wed Jul 05, 2006 6:22 am

thanks for the response

yes, the temperature conditions holds tru if the experiment was conducted as by Muller, carring each reaction in a seperate water bath with predefined temp....

and in the present robotic apparatus the concentration are major reason .


thanks again for the response
B.V.Krishna Rao
Research Associate
Shantha Biotech, India

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