Hi Theodore,

Nothing is black/white in biology, so it is with the dissociation of primers. Tm means that at this temperature, 50% of the primer molecules are bound to the dna, and the other 50% are dissolved. Cave, the Tm temperature is only based on computer algorithm, and can vary certain degree under real circumstances. In your case, setting the Ta to 60C means, that roughly 40-20% of the primers might bind. In this case you have two possibilities:

When the probability of primer dimers is very low, you can increase specifity by increasing the concentration of primers and increasing the Ta, to obtian the same product yield.

In contrast, when you experience that primer dimers form easily, it is the other way around; lower Ta, lower amount of primers.

Of course this simple scheme has its limits, I just wanted to point out, that there is no all-or-none principle in PCR.

regards

Hi Theodore,

Nothing is black/white in biology, so it is with the dissociation of primers. Tm means that at this temperature, 50% of the primer molecules are bound to the dna, and the other 50% are dissolved. Cave, the Tm temperature is only based on computer algorithm, and can vary certain degree under real circumstances. In your case, setting the Ta to 60C means, that roughly 40-20% of the primers might bind. In this case you have two possibilities:

When the probability of primer dimers is very low, you can increase specifity by increasing the concentration of primers and increasing the Ta, to obtian the same product yield.

In contrast, when you experience that primer dimers form easily, it is the other way around; lower Ta, lower amount of primers.

Of course this simple scheme has its limits, I just wanted to point out, that there is no all-or-none principle in PCR.

regards

Hi Theodore,

Olivier is right, it is a difficult question. Primers' melting temperatures are affected by various factors. The melting temperatures of the same primer pairs may differ in different tubes with the same templates depending on the primer and salt concentrations, etc. Moreover, some PCR additives i.e. DMSO may affect the melting temperatures. It has been reported that 10% DMSO can decrease the melting temperature by 5.5-6.0 Celsius.

Higher annealing temperatures generally increase specificity but may decrease sensitivity of PCR.

Regards

Hi Theodore

I also agree with Oliver regarding the 'Calculated Tm' and 'Actual Tm'....the Tm actually in the PCR reaction depends upon several factors such as, buffer composition, metal ion concentrations, PCR additives (if any), Denaturants (if any) etc. and in general the 'working Tm' are higher than ' calulated Tm' and they work fine....

bye

Try using http://bioinfo.weizmann.ac.il/blocks/oligo_melt.html , it accesses several tools to give an overview of different approached for Tm calculation. I've found out over the years that in PCR and qPCR the actual temperature is usually precisely what the output is for Breslauer on PRIMER3.

And if you want to go extremely high, SantaLucia's NAR Tm can give a good idea of he upper limit they will bind.

Several tools for reliability of results usually calculate the Tm and then give something 4-5 degree Celsius lower - because if the actual Tm is correct, a lower temperature will still give you hybridization even if you add Betaine or DMSO. It is basically a compromise of feasibility they sneak in.

Whether Tm is higher or lower than Ta, is perhaps a secondary question to you and more important is whether you get QPCR amplification at Ta 60. If not go back to what you standarized and run three step PCR using Ta 54 and extension at 60.Well perhaps you would be knowing already but still I would mention that in run Method you can always customise the QPCR program as per my experience on ABI 7000 and ABI 7500 fast. If it helps..........

Good Day

Hi theoder

i too tried annealing temperature above the higher tm of primer. As my experience it is better to try always with 5 degree temp lower and higher than the avg tm of primers. We cant say that it should not be always lower than the lower tm. Product may come at 3 to 4 diff temperatures. So no need of worrying about higher temp or lower temp, only thing is we should get a good amplifications. Primer designing conditions also shows effect on your primer annealing temp. Hv a nice day

Theodore,

Evey algorithm used to predict Tm and Ta of primers use different buffer conditions. What you need to pay attention to is the salt concentrations, particularly Na+ and Mg++. As a rule of thumb, higher the salt concentrations, more relaxed the amplification conditions - meaning that the same Tm can yield more non-specific amplifications at higher salt concentrations. The specificity of primers also depend on the PCR buffer from each vendor. If you don't know what the buffer composition is, the best way forward is to try a range from 5 degrees below to 5 or more degrees above the predicted Ta. Choose the highest Tm that yields the most specific product. As a general rule, the Ta is usually 5 degrees below the Tm. I always check my primers using regular PCR, run the product on agarose gels to make sure that they amplify the right size fragment before going for qPCR. In my experience, specific detectable product on an agarose gel will yield very good qPCR product.

Hello Theodore,

Have you ever ran a gel? I suggest run a gel using the gradient temp templates and your 60C qPCR product. Check if you got the right product (no double bands), and right size. After this, you can decide which Ta will work best, and which Tm you will get for your product. Good luck!

In theory, the Tm is the temperature where half of the DNA is still double stranded and half has melted away. So at the Tm, half of the potential DNA duplexes would be intact. But as many here have said, algorithms for Tm calculation give widely variable results and the only reliable test of the best anneal temp is the kind of optimization that you did. Did you show your lab manager the results of your gradient PCR experiment? Is there any reason to expect that the PCR conditions during your optimization test are greatly different than the PCR conditions during the qPCR? And what was the efficiency of amplification during the qPCR run when the Ta was set at 60? (If highly efficient, the fluorescent signal should double or very nearly double with every cycle of PCR during the exponential phase.) If this anneal temp (60) is too high and causes the PCR reaction to be inefficient, then your qPCR data will not be accurately quantitative, since the delta Ct method assumes that the efficiencies of the reference and test PCRs are high and equal. On the other hand, if annealing at this temp (60) still gave efficient amplification during the qPCR and the product was specific (sharp single dissociation peak if Sybr green, with gel check of product) then you have no worries.

Proc Natl Acad Sci U S A. 1986 Jun;83(11):3746-50.

Predicting DNA duplex stability from the base sequence.

Breslauer KJ, Frank R, Blöcker H, Marky LA. Thermodynamics is a nice issue :)

There is no doubt a about Ta = Tm - 5 !!!

one of the many calculators - http://www.finnzymes.fi/tm_determination.html

nice day

qPCR requires less non-specific polymerization and primer dimer than normal PCR, A higher annealing temperature increases amplification specificity by discriminating against incorrectly annealed primers and reducing mis-extensioin of incorrect nucleotides.

Hi every one

The Ta is the optimum annealing temperature. Yes it is usually around 5 degrees below the Tm (although this varies depending the composition of primer and length of primer and I would always advise a temperature gradient PCR to determine the actual optimum annealing. 

You can read more about that

https://www.idtdna.com/pages/support/technical-vault/faq/browse-all-questions-answers/how-do-you-calculate-the-annealing-temperature-for-pcr-

Cam anybody explain Why Tm varies for same oligonucleotides. I purchased primers; (sequence reported in the literature) which has Tm 82 and 83oC. for these promers Ta will be ~78oC. However, in the the literature people have used annealing temperature 63, 64oC. That means Tm for the same set of primers would be 68-70oC. Why such a difference occurs. And what should be Ta for our set of primers.

because the -5 rule is just an approximation that sometimes works others does not. Rychlik formula works much better, gradient PCR testing even better