"Primer Melting Temperature (Tm) by definition is the temperature at which one-half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability. Primers with melting temperatures in the range of 52-58 oC generally produce the best results."
During initial denaturation at 95C and denaturation at 95C DNA is completely becomes single stranded. At 55-65C they anneal to the DNA at 72 Taq is most active and extends. Annealing temp should be 5C below Tm. What is half of the DNA duplex will dissociate at 52C mean? Is Tm and annealing temp of primers are different?
Say at Tm 50% of the primers bind then what is annealing temp mean?
Tm describes the situation where there is limited amount of template ssDNA and a large excess of primer.The Tm is the temperature at which the equilibrium of free primer and free template is in equilibrium with primer bound to target dna and the bound primer is binding and falling off(melting) so that 50 % of the template has bound primer. as soon as this happens in pcr the enzyme extends the bound primer so the equilibrium has to be maintained by more primer binding. The Ta is a more practical temperature and it is the temperature used in pcr so that binding occurs and at the most efficient and specific rate. Higher Ta means more specificity but less binding so ensures a clean product but may need a few more cycles. Ta can be up to 12C above the Tm but is most often about 2-5C above the Tm. The Tm can change with reagent concentration .pH and salt concentration. There is always a huge excess of primer in pcr as after about 30 cycles there may be 10exp9 times more product than the initial concentration all of which has one primer molecule incorporated so the 50% in your definition refers to the 50% that bind to template and has very little to do with initial primer concentration
Dear Xin Pin,
The definition of Tm you give is correct. Also, the usual recommendation for annealing is indeed 5 °C above Tm. However, there are probably half a dozen different algorithms to calculate Tm. For my part, I don't bother and use the Thermo Fisher Tm calculator (you can Google the exact address of the web site) to design my primers and PCR protocols
Tm describes the situation where there is limited amount of template ssDNA and a large excess of primer.The Tm is the temperature at which the equilibrium of free primer and free template is in equilibrium with primer bound to target dna and the bound primer is binding and falling off(melting) so that 50 % of the template has bound primer. as soon as this happens in pcr the enzyme extends the bound primer so the equilibrium has to be maintained by more primer binding. The Ta is a more practical temperature and it is the temperature used in pcr so that binding occurs and at the most efficient and specific rate. Higher Ta means more specificity but less binding so ensures a clean product but may need a few more cycles. Ta can be up to 12C above the Tm but is most often about 2-5C above the Tm. The Tm can change with reagent concentration .pH and salt concentration. There is always a huge excess of primer in pcr as after about 30 cycles there may be 10exp9 times more product than the initial concentration all of which has one primer molecule incorporated so the 50% in your definition refers to the 50% that bind to template and has very little to do with initial primer concentration
"Higher Ta means more specificity but less binding"
less binding due to melting? if it's specific then again why less binding? the temperature was good enough to bind specific region so why less binding?
"one primer molecule incorporated"
and this incorporated primers also gets cleaved by Taq 5'-----3' exonuclease activity is that correct or the Taq stars extending it from 3' OH of primer?
Less binding both in the sense that binding to similar but not identical sites happens much less but also in that the binding and melting of primer onto and off of its template target is an equilibrium so at higher temperatures the primer melts off quicker so the enzyme has less chance to stick,stabilise the double strand to single strand junction and start extending the product. After the extension has started the double strand is longer so much more stable and extension happens at more than 100 bases a second so you will get product very quickly. Also the huge excess of primer makes sure that there is always some primer trying to anneal so the reaction works even if the primer melts off quickly because another primer is around to anneal. Processivity happens from the 3' end of the primer and all pcr product has the primers incorporated into the product. There is no cleavage of primer. This is why you can incorporate restriction sites or any wanted sequence on the 5' end of your primers without interfering with the amplification of the product
Sorry for the late reply I haven't received notification about your comment.
"stabilize the double strand to single strand junction"
During annealing doesn't the DNA duplex also get annealed? no room for primers to bind?
"Primer Melting Temperature (Tm) by definition is the temperature at which one-half of the DNA duplex will dissociate to become single stranded and indicates the duplex stability."
Half the DNA gets dissociated fine what if our seq is within the other half that is not dissociated?
We are calculating primers Tm not DNA Tm? (or it's like at primers Tm half of DNA duplex is still unbound?) if that is the case at 5C below Tm primers bind so does DNA?
and during extension don't the primer extented seq melt off at 72C 10 min hampering Taq synthesis.
So primers get incorporated. But taq has 5' nuclease activity in qPCR probe gets degraded by 5' flap endonuclease activity. So I was thinking same happens in end point PCR. In that case, there shouldn't be any amplification I'm confused.
Although annealing temp should be 5C below Tm many take the avg of Tm's and it still works?
During annealing doesn't the DNA duplex also get annealed? no room for primers to bind?
Yes the other strand of the template always tries to re anneal and at a higher temperature than the primer but the huge molar excess of primer means that usually a lot of primer sticks and the reaction works. Sometimes with high GC templates the template reanneals too well and the pcr does not work. Then the template sequence has to be kept melted for longer using DMSO or betaine. Higher annealing temperature primers always helps.
Half the DNA gets dissociated fine what if our seq is within the other half that is not dissociated?
I think that you are misunderstanding here. Half of the dna means , It refers to one half of the mass of dna EG if you have 1ug then one half is 0.5ug not the first or second part of the sequence of the dna
We are calculating primers Tm not DNA Tm? (or it's like at primers Tm half of DNA duplex is still unbound?) if that is the case at 5C below Tm primers bind so does DNA?
Yes when 2 molecules have greatly different Tm s then you only need to worry about the Tm of the lower annealing molecule...in this case the primer. At %c below the template will re anneal but the greater amount of primer means that much of it will anneal and work.
and during extension don't the primer extented seq melt off at 72C 10 min hampering Taq synthesis.
No the enzyme works at all temperatures and extend at more than 100 bases per second. With annealing and the time taken to get to 72 the primer has extended by hundreds of bases so the duplex cannot melt at 72
Although annealing temp should be 5C below Tm many take the avg of Tm's and it still works?
No PCR needs 2 primers working so both must anneal. The only cetrtain way is to run a gradient pcr at different annealing temperatures because Ta is variable and primer sets will often work well at up to 10c above the Tm whatever the theory says.
I think that 5' to 3' exonuclease activity is not relevant because there is no extension from the 5'end of each primer at the ends of the pcr product because all extension occurs 3' to 5' but someone else will give a better answer to this aspect of the question I hope
Does Tm have to be 5C below or 5C above? or is it +/- 5C I read it as 5C below Tm. And if I understand correctly at Tm primers and template are kind of in equilibrium although primers binding to the template yet quite unstable at Tm? (binding and falling off) So at temp above or below make more stable binding of primers to the template.
"Yes the other strand of the template always tries to re anneal and at a higher temperature than the primer".
I don't get the logic as the temp increases strands tend to separate so how does going 5C above Tm helps primers binding to the template.
Alright if I apply your logic "the other strand of the template always tries to re anneal and at a higher temperature than the primer".
At 95C two strands are completely denaturated and while moving from 95C to 58C by the time it reaches to 58C DNA must have been reannealed?
"Yes when 2 molecules have greatly different Tm s then you only need to worry about the Tm of the lower annealing molecule".
Can you try explaining this with an example? Say primers have Tm 62C and DNA Tm is 82C? if 82C is wrong what is the range of Tm for DNA? So at 87C DNA anneals? since temp rapidly moves away there isn't much time spent at 87C so DNA doesn't anneal as much. But as temp decreases DNA should bind? irrespective of DNA Tm.
"Tm of the lower annealing molecule" what is lower annealing and how are primers lower annealing molecules?
Sorry Xin Pin I never saw these last questions. Much time has passed. Do you still need answers to the last questions or are you happy with the technique now?
ok some thoughts
Does Tm have to be 5C below or 5C above
Pcr runs at different temperatures with different specificity depending on where the primers anneal. At 5c below you can be sure the pcr will work but your primers will find other regions of the genome to anneal to so you will get many products forms as well as your wanted product. The increase in temperature above Tm destabilises the primer annealing so if your primer is annealing to sequences with only 90% homology at low temperatures the higher temperature ensures that only perfect homology between primer and target dna is permissible so you only get the perfect match product.
Alright if I apply your logic "the other strand of the template always tries to re anneal and at a higher temperature than the primer".
Yes the other strand is always trying to re anneal which is why longer ( higher annealing) primers work better but annealing and melting are equilibrium situations. It is better to have a fast fall in temperature to annealing to minimise self annealing but the huge excess of primer will allow a lot of primer to anneal because in the early cycles there are much fewer molecules of template so they find each other with difficulty but the huge number of primer molecules anneal quickly . In the latter stages of pcr self annealing is more of a problem. Remember also there is nothing special about 95c and your product may well melt completely at 85c so the cooling time in real terms may be quite short
"Tm of the lower annealing molecule" what is lower annealing and how are primers lower annealing molecules?
What is important here is that both primers must anneal for logarithmic amplification of a product so the pcr conditions must allow the primer with the lowest annealing temperature to anneal so with primers with Ta of 60c and 50c you should either lengthen the 50c primer so it anneals at 60c ( ideal) or set the annealing temperature of the pcr cycler to 50c so that both primers can anneal, If you do this then the 60c primer can possibly bind to sequences with only 80% homology with itself and the pcr may become dirty with amplified bands that you do not want
Tm or melting temperature is a certain temperature which half of double-strand DNAs turn into singal strand DNAs (half of them are still dsDNAs). In other words, the Temperature of Melting (Tm) is defined as the temperature at which 50% of double stranded DNA is changed to single-standard DNA. Tm is basically depends on GC conent.
In PCR experiments, GC content of primers are used to predict their annealing temperature to the template DNA. A higher GC content level indicates a higher melting temperature.
Tm is, in fact, a critical parameter to consider when designing and performing molecular biology experiments, including PCR and qPCR. During the annealing phase of PCR, the reaction temperature needs to be sufficiently low to allow both forward and reverse primers to bind to the template. Both primers in PCR should be chosen to have a similar Tm; it is recommended that an annealing temperature should be 5–7°C below the lowest primer Tm.
The formula for primer Tm calculation: Tm = 4(G + C) + 2(A + T)=°C
Thanks for Paul, Xin and Mehrada for their nice and interesting discussion, especially for our postgrag. students.
Najim Al-Masoudi
Konstanz, Germany
web: www.al-masoudi.de
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