I use the program Primer3 available at http://primer3.ut.ee/ for designing my primers. The program allows you to introduce your own primers and check for the stability. Primer design can be done manually or using many different programs available for free or in the market, having all similar results. At the end, because primer behavior depends on the thermodynamical conditions in your PCR (salt concentrations are very important), to obtain good results are an empirical question. But if I have degenerate primers I use the rule used by Boris to check for my maximum and minimum melting temperatures.

Hi,

here is the formula's for calculation of the Tm (melting temperature in °C) for a primer of 20 nucleotides or less.

Tm: 4 x (G+C) + 2 x (A+T)

For longer one :

[4 x (G+C) + 2 x (A+T)]x(1+[(N-20/20)]=Tm (were N is te number of nucleotide)

By this calculation you can choise between diffrent primer, their compatibility a te same Tm.

I had used those formula many times and it works very well for discrimation of primers.

Hope it will help

I wouldn't have thought it was that simple.

If 100% of your primer bases match 100% of your target and you set the stringency conditions of your PCR up right then you should have almost complete specificity (barring things like primer dimers).

Why are you trying to work this out?

I use the program Primer3 available at http://primer3.ut.ee/ for designing my primers. The program allows you to introduce your own primers and check for the stability. Primer design can be done manually or using many different programs available for free or in the market, having all similar results. At the end, because primer behavior depends on the thermodynamical conditions in your PCR (salt concentrations are very important), to obtain good results are an empirical question. But if I have degenerate primers I use the rule used by Boris to check for my maximum and minimum melting temperatures.

Hi Andrew, I understand you are trying to measure the specificity of a primer sequence to its target... there are a couple of options out there:

1) The S score which is a measure of the similarity of the query to the sequence

shown, and

2) The E−value which is a measure of the reliability of the S score.

The definition of the E−value is: The probability due to chance, that

there is another alignment with a similarity greater than the given S

score.

E−value Equation

The actual equation is E=Kmn(e−λS)

The parameters K and λ represent natural scales for the

search space and the scoring system respectively.

The rest of the equation represents the size of the query (m), the

size of the database (n), and of course the S score.

for additional details and reference see the attached link to document

Hope this help. Cheers!

http://biophys.chem.columbia.edu/biophys/evalue.shtml.pdf

I think I perhaps did not phrase my question clearly, my apologies.

I was not looking for an equation to calculate melting temperature.

My primer bases do not match 100% of my target because they are derived from best conserved region homology of closest related species in an area never before sequenced in this animal, and they include wobbles to cover variance in a few nucleotides.

In the very long gene I am sequencing I have at least 11 conserved areas that may be where possible primers could be allocated in order to sequence variable areas in between. The conserved areas can be up to 30 nucleotides in length with some minor wobbles dispersed throughout so that no 20 nucleotide selection I might make has less than 3 wobbles.

So the reason I am interested in working this out, is while it perhaps may come as tacit knowledge to others it is new to me; which is why I am looking to find or create an equation, so that once I have my known length, known Tm, known CG/AT ratio, and known wobble density of wobbles across GC/AT boundaries, of each selected possible primer, I can then form an equation which would then relate the specificity of each choice, and could even be mapped graphically.

There may not be an equation for this, although one would be useful.

Andrew,

From a 'problem solving' perspective... I mean trying to put you on wheels toward degenerate primers to amplify your unknown gene sequence fragment, you may try to design primers starting from the degenerate sequences you already have in possession using a couple of software that are of free access in the WEB. One is CODEHOP (not very friendly to use) and the second is PRIMACLADE (friendly). This can give you a temporary peace of mind while you develop the algorithm you intend for your purpose which can demand some time if having the intention to validate it in the end. Hope this help... Cheers!

http://primaclade.org/cgi-bin/primaclade.cgi

Andrew,

In 2001 I created a program called Virtual PCR (https://www.researchgate.net/publication/12094037_Virtual_PCR) which I think does the kind of calculations you need. It does it on any (genomic) sequence database. It was upgraded in 2003 or so and later mentioned in a paper together with some ideas of how to speed it up on FPGAs (https://www.researchgate.net/publication/213744795_Genomic_PCR_simulation_with_hardware-accelerated_approximate_sequence_matching) (https://www.researchgate.net/publication/8966263_PRIMEX_rapid_identification_of_oligonucleotide_matches_in_whole_genomes).

Article Virtual PCR

Conference Paper Genomic PCR Simulation With Hardware-Accelerated Approximate...

Article PRIMEX: Rapid identification of oligonucleotide matches in w...

sorry, clicked the wrong button...I do not have it running anywhere, but if you are or have an IT person around, you may be able to install it, see the attached files. You may also need themex-0.99 program (PRIMEX), that I have on ResearchGate, or run it with BLAST (not recommended). It is not very user friendly to install, I am sorry, but it does work. I can help you if necessary.

Unfortunately if You want to know, You have to measure the melting temp. of a primer on your own. There are no good predictors, because the sequence / neighbourhood effects cannot be precisely calculated. In my oppinion you can obtain the best available today, but a rough calculation - using primer3.

Try this free software at [http://www.generunner.net/]. Copy paste your whole gene sequence and then Ctrl+F the primer seq you have. There u can choose to detect your various specificity reqs e.g. dimer formation, loop formation, internal looping etc. I do not think any formula is going to help in detecting specificity online. In fact you need to ask for parameters in your question not the formula. Formula gives you the Tm value only infact. I hope this helps.

Hello everybody!

I gave this a little bit more thought yesterday and felt like sharing some of it with the board. Andrew mentioned "specificity". To me, this translates into the following question: "How specific is the primer going to be for the intended target? Is it going to bind somewhere else?". As a result, it also becomes a question of how efficient the PCR amplification with these primers will to be? Am I going to amplify the intended region, or perhaps also some other regions of the template? While anything based on the nearest neighbor method, like the original SantaLucia calculations, Primer3 Tm calculatios or perhaps Unafold from Zuker et al. will give reasonable Tm estimates, or even examine secondary structure formation, design a 3' clamp and what not - even then - it may not tell me much about specificity. If the template happens to be genomic DNA, we need some calculation of where else the primers could bind. That's why I mentioned Virtual PCR. Of course, some of the other programs can also address this issue, for example by examining how likely the primers are to match a repetitive sequence, perhaps by comparing it to the latest RepeatMasker database. I lost track of these capabilities lately, maybe Primer3 does this, maybe not, I don't know. Still, low copy dispesed repeats are probably not well covered by RepeatMasker.

The biological situation is very important for addressing questions of "specificity". How complex is the genome of the organism you are working with? Is it a bacteria with a genome size of a few million bp or a tetraploid plant? Is the gene you are trying to amplify relatively unique or is it part of a gene family with many similar genes in the same genome? Calculations of primer melting temperatures may be useful for picking an initial set of conditions for PCR reaction set-up, but whether your amplification product is "specific" depends on many other factors. If you are trying to sequence a cDNA, it's possible that you may need to resort to the cloning approach.

One of the best algorithm I know to calculate Tm of primers can be found and tried at:

http://www.biophp.org/minitools/melting_temperature/

The demo:

http://www.biophp.org/minitools/melting_temperature/demo.php

I just found this question in this Q&A section: https://www.researchgate.net/post/Does_anybody_know_of_a_good_alternative_to_iCODEHOP_as_a_tool_for_designing_degenerate_primers?ch=reg&cp=re221_b_p33&pli=1&loginT=JoYOyxafOldfGJFhEO0nmp4UbapbeXJdKLasQgTIUmE. It may help in your problem