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PCR: Low Primer Annealing Temperature Explanation?

Genetics as it applies to evolution, molecular biology, and medical aspects.

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PCR: Low Primer Annealing Temperature Explanation?

Postby AciesEdge » Sat Oct 06, 2012 10:49 am

Hi everyone,

I've just got a quick question regarding a specific step within a polymerase chain reaction:
In the PCR, I understand that the primers have a specific temperature in which they best function and that a temperature which is too low will cause them to anneal to non-specific locations of the DNA molecule.

However, I was a little confused behind the reason as to why this happened. I've heard some explanations/theories from other students, but I'd like to have a verified, solid answer, which is why I thought I'd consult this forum.

I do apologise if this is in the wrong section or if the question's been asked before. I'm just in a bit of a rush at the moment, but thank you in advanced for your help!
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Postby Cat » Sat Oct 06, 2012 1:26 pm

That temperature is called Tm which stands for melting temperature. Tm is a temperature at which 50% of primer is free and 50% bound to the template. Since each mismatch during annealing of the primer lowers the melting temperature of such a pair, non-specific amplification is lowered by increasing the temperature. Normally, you start with Tm as your annealing temp and if you still get non-specific amplification, you do several runs increasing the temp and find one at which your non-specific product disappears...
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Re: PCR: Low Primer Annealing Temperature Explanation?

Postby wbla3335 » Sat Oct 06, 2012 2:30 pm

A bit more detail. Three things:

1. Strands of DNA stick to each other through hydrogen bonding.
2. Hydrogen bonding will occur whenever and wherever conditions allow it.
3. The molecular world is a rather chaotic place, with molecules jostling about (Brownian motion) and with hydrogen bonds continuously forming and breaking at rates and frequencies determined by the amount of thermal energy (temperature).

To understand what happens in a PCR reaction, it is helpful to view the reaction mixture as populations of molecules. Each primer is a population of molecules, and each strand of the template DNA is a population of molecules (after melting of the template DNA). Any one molecule of primer may stick anywhere on a molecule of template DNA whenever hydrogen bonds can form. The duration of the annealing will depend on the temperature and on the number of hydrogen bonds that form between the primer and template molecules. The optimal annealing temperature is that where the primer molecule forms the maximal number of hydrogen bonds with the template molecule to produce a maximally stable association. Again, think of the reaction mixture as a chaotic mix of populations of molecules. Associations between primer molecules and template molecules (and also between different primer molecules and different template molecules) are constantly forming and breaking. Even individual perfect matches can dissociate at the optimal annealing temperature, but within the population of primer molecules, a sufficient number of associations will endure long enough for molecules of polymerase to latch on and begin to extend the primer molecule. Even though the polymerase molecules function optimally at higher temperatures, they will start to do their thing at the lower annealing temperature. Submaximal associations between primer molecules and template molecules can also get extended, but a competition then begins between the perfect matches and the imperfect matches. The perfect matches will ultimately win, because they are more numerous within the population. At the end of the PCR programme, a PCR tube will contain the desired product (hopefully) but also all kinds of crap that were the losers of the innumerable competitions that were waged during the programme (mostly during the early cycles).

A lower annealing temperature will increase the number and duration of mismatched associations, thereby increasing the probability that mismatched primers will get extended. The ensuing competitions will be more numerous and will produce less target product and more crap.

I've always found it helpful to understand the molecular world if I try to imagine myself being in that world and witnessing what's happening.
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Re: PCR: Low Primer Annealing Temperature Explanation?

Postby AciesEdge » Sun Oct 07, 2012 1:48 pm

Hey guys, thanks for giving me a hand :D

I hope it's okay, but I'm using the explanation that you've given me in combination with information from the internet to help me answer a question for my practical report. This was my first time performing a PCR in Uni, and so there are some concepts and details regarding its process I'm not familiar with. So I've just included my answer to my practical question below and was wondering anyone could check to see if my understanding is correct.

2. What would be the expected result if the annealing temperature of a PCR was too low for the primers in the reaction? Explain
My Answer:
In cases in which the annealing temperature of a PCR is too low for optimal primer function, annealing may occur at non-specific or mismatched sites compared to the primer’s true target. This occurs as at sufficient energy generated by the temperature of the mixture, hydrogen bonding between the primer and template DNA strand is at an optimal level in which the primer and DNA strand are able to form maximally stable associations. However, at lower temperatures, there is overall less energy in which the primers are subjected to, resulting in increased partial annealing and a reduced potential to form the aforementioned maximally stable associations. Consequently, without optimal temperature, the altered primer function may lead to non-specific amplification and overall reduce the quantity of product yielded.
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Postby Cat » Sun Oct 07, 2012 4:16 pm

Your answer is difficult to read and confusing. Some things jump out. Instead of saying "annealing temperature of a PCR is too low for optimal primer function" you should say "annealing temperature below optimal". Also in the last sentence "reduce the quantity of product yielded" should say "reduce the quality of product yielded".

I would say this instead:

"Using a low annealing temperature would most likely result in amplification of undesired sequences in addition to that of the target. This is due to generation of amplicons from non-specific primer annealing when this step is performed at temperature insufficient to melt such mismatched pairs."
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