PCR

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Sucheta
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PCR

Post by Sucheta » Wed Feb 28, 2007 11:28 am

In PCR technique, during the annealing stage, why the parent DNA templates do not anneal where as primers do in each cycle?

Vagabond
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Post by Vagabond » Wed Feb 28, 2007 12:51 pm

It has to do with the amount and shear size of he parent molecule. The primers are small and present in way over abundance. The parent molecule is huge compared to the primer (and all basepairs have to match up). I recall that the textbook Genes (don’t know what number they are on now) has a good section on DNA annealing times. (I believe it was 5 or 6 that had a nice graph).

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Sucheta
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Post by Sucheta » Fri Mar 02, 2007 7:10 am

The reason seems logical.

But does temperature plays any role in annealing?
Is it possible that the oligonucleotide primer anneal at a pre-set temperature which is different from the temperature required to anneal the parent template?

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Post by Vagabond » Fri Mar 02, 2007 1:09 pm

Yes very good observation. Temperature does play a role in annealing. Primers of to high a GC ratio or long primers will have higher annealing temps. I will try an post back on with more details but I have a few meeting today.
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dipjyoti
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Post by dipjyoti » Fri Mar 02, 2007 5:06 pm

The specific complementary association due to H2 bonding of single-stranded nucleic acids is referred to as "annealing": two complementary sequences will form hydrogen bonds between their complementary bases (G to C, and A to T or U) and form a stable double-stranded, anti-parallel "hybrid" molecule. 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. Additionally, if the NA is heated in buffers of ionic strength lower than 150mM NaCl, the melting temperature is generally less than 100oC - which is why PCR works with denaturing temperatures of 91-97oC.

Tm = 4(G + C) + 2(A + T)oC.
Attachments
pcropt1.gif
Plasmid and biopsy sample DNA templates were amplified at different annealing temperatures as shown: note that while plasmid is amplified from 37 to 55oC, HPV DNA is only specifically amplified at 50oC.

Reff: Dept Molecular & Cell Biology
University

scmesser
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Post by scmesser » Sat Mar 10, 2007 7:50 pm

need my 5 posts - 3, sorry!

hadis
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Post by hadis » Wed Mar 10, 2010 7:41 pm

hi, i have a problem , can you help me in this issue? :|
i design a primer in blast , it was specific when i checked it in blast but when i used it, i had nonspecific band , what can i do now? if i use high temp. for annealing ,will my result improve? :!: :?:
thanks

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JackBean
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Post by JackBean » Thu Mar 11, 2010 8:31 am

Did you also check autokomplementarity?
You could try to change the temperature, you could try 2-step cycling, adding additives (like DMSO, depends on your polymerase) or change the polymerase
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

aniroc
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Post by aniroc » Fri May 28, 2010 1:24 pm

Hi! I'm new to this and to PCRs :-(- and desperate to find out if my using only 1 ul of homemade Taq instead of 1,5 (like the "recipe" said) will affect much ( and how) my PCR ! It will be done in about 15min, but I would very much like to know if I'll have to come back in the week-end and repeat it ... Thanks!
AniRock

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JackBean
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Post by JackBean » Tue Jun 01, 2010 8:29 am

That depends on activity of your Pol, if is it highly active even in the 1 ul than it's for sure fine, but if you have only low activity than even 5 ul may not be enough ;)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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