I have a couple of primer sets and am interested in calculating their theoretical annealing temperature before the actual optimization. I understand we can use +-5C of Tm gradient and find the exact Ta. But I wonder whether anybody has ever used an equation which can possibly tells us anything about Ta of a given primers. I found this equation on the web
Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 25
But I don't necessarily know how to find the Tm of product or it gives a temperature above 1000Cs.
Please advise me. Thanks
Dear Harindra,
Two standard approximation calculations are used.
For sequences less than 14 nucleotides the formula is:
Tm= (wA + xT) X 2 + (yG + zC) X 4
And for sequences longer than 13 nucleotides, the equation used is
Tm= 64.9 + 41 X (yG + zC - 16.4) / (wA + xT + yG + zC)
where w,x,y,z are the number of the bases A,T,G,C in the sequence respectively.
Hope these may come to your help
The annealing temperature (Ta) chosen for PCR relies directly on length and composition of the primers. Generally, you should use an annealing temperature about 5°C below the Tm of your primers. The optimal annealing temperature (Ta Opt) for any given primer pair on a particular target can be calculated as follows: Ta Opt = 0.3 x(Tm of primer) + 0.7 x(Tm of product) - 25; where Tm of primer is the melting temperature of the less stable primer-template pair, and Tm of product is the melting temperature of the PCR product.
One consequence of having too low a Ta is that one or both primers will anneal to sequences other than the intended target, as internal single-base mismatches or partial annealing may be tolerated. This can lead to nonspecific PCR amplification and will consequently reduce the yield of the desired product. Conversely, too high a Ta may reduce reaction efficiency, as the likelihood of primer annealing is reduced significantly. Optimal annealing temperatures will result in the highest product yield with the correct amplicon.
I was taught the old rule of thumb while I was a student and it has worked for me thus far: Tm= 2*(# of A or T) + 4* (# of G or C) - 5* (1+ # of mismatches between primer and template). I did a lot of PCR based mutagenesis, which is why the mismatches are there.
I believe if you design primers in such a way that they have 20 metes and equal number of nucleotides, then you can use the equation: (A:T) equal 5
(C:G) equal 10 for each nucleotide i.e annealing temp equal 60 C
Most primer desing use below formula in our Lab. :
Tm= (A +T) number X 2 C+ (G + C) number X 4 C
Most desing primers equal 60 C according above formula.
The biotecnology company will provide more near accurately Tm after synthesis.
Finally, the best annealing temp was decided using PCR machine.
Probably you were not the one to design the primer because any software’s or websites used to design the primer would give you the theoretical annealing temperature. In the case of taking it from the literature, you could use the following website to have an estimate. I recommend using UCSC In-Silico PCR, Primer blast or PCR Primer Stats. PCR Primer Stats would additionally describe the properties of each primer including GC content (%) and PCR suitability tests such as self-annealing, hairpin formation, GC clamp etc.
http://genome.ucsc.edu/cgi-bin/hgPcr
http://www.ncbi.nlm.nih.gov/tools/primer-blast/
http://www.bioinformatics.org/sms2/pcr_primer_stats.html
Dear Prof Arthur,
Thank you for your explanation. I understand. Since I'm not a molecular biology person, I wanted to have a rough idea about the possible Ta for primers. So I can literately stay within the range during the optimisation.
What would you suggest when it comes to temperature gradient? (Tm +- 5C?)
Dear Prof Artur,
Thanks, I'm certain that would be a help. Just one thing to get clarified.
Am I supposed to do the temperature gradient first and then (if I get any band on the gel) adjust the Mg concentration or the other way around?
Thanks
There are several methods to compute a theoretical Tm
try
http://www.ebi.ac.uk/compneur-srv/melting/ probably the best
http://www.mybiosoftware.com/pcr-primer-design/6607
or
http://melolab.org/dnaMATE/tm-pred.html very simple
If you have both target and non target (that you don want to amplify) take a look at
http://bioinfo.unice.fr/softwares/ohm/
finally, a list of tools
http://omictools.com/primer-quality/
Its depends in general on the GC%, and Tm of the primer further more the length of amplicon
Ta= Tm (minimum)+ lin L
where L is the length of PCR fragment
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