The stability of the 3' complementarity does not have to be very high to get dimers, because the primers are in high concentration (think Moles not micrograms) in the first rounds, relative to the target template concentration.  A two base complementarity is enough.

Hello Caner. I concur with above answers. High GC content particularly at the ends of primers can lead to annealing extension and primer dimer products. Ultimately what matters is not the propensity to form but how stable these duplexes are under your reaction conditions such that self annealing leads to extension as described by the first speaker. To predict self annealing and in particular whether such duplexes are stable and could give rise to primer dimers there are various programs that will predict such structures. To that end find a link to a word document prepared by on primer design including links to programs you can use to screen for intra complimentarity I.e hair pin loops and inter complimentarity culminating in primer dimers potentially. 

https://www.dropbox.com/s/m4vfq4wjscnkm7w/PRIMER%20DESIGN%20document_updated%200414.docx?dl=0

Primer dimer formation is very unpredictable. As the replies above point out, there are conditions, which do favour dimerization. However,  I have designed thousands of primers by eye and using software (primer3, netprimer, vectorNTI). Primers that look perfect and are predicted to not dimerize or form hairpins often work well, however primers that are predicted to dimerize extensively and hairpin often also perform well. Don't spend a lot of time trying to design the perfect primer if you are struggling, many that look bad on paper often work as well as, if not better, than those that don`t

Good luck

I agree with the last commentator. The sorts of algorithms used in programs such as IDT oligo are just predictions. They don't always bear out in reality. One thermodynamic parameter is delta G which is the thermodynamic free energy of a structure. This allows you to make predictions with regard to how stable in actuality a structure will be and thus it's real propensity to cause such problems as primer dimers. See the link from my word document referred to in my last answer

In addition it is possible to make the reaction TD conditions more denaturing: namely by adding 1or 2 degrees to the denaturation cycle; add in a substance called Betaine at 1M final concentration and preceded the TD phase of the PCR reaction with a single denaturing cycle of 96c for 2-5 min

i agree with all of the above but in practical terms primer dimer is only a problem if the temperature of the reaction falls low enough for a few bases ot he primer to anneal.Using hot start enzymes means that the reaction mixture never drops below the full annealing temperature of the primers so usually solves any possible primer dimer problems as the enzyme is only activated at the first denaturation step of the pcr

Thank you very much to you all for your informative responses. 

However the exact problem I confront for the several times is primer dimers and some nonspesific bands just appear in a position lower than the marker (3000-100bp) however i could not get the desired band I would like to see even appearance of primer dimer and nonspecific bands are very shiny and thick. As I said, I am using touchdown PCR by beginning from anneal temperature which is choosen to be 3 0C higher than optimal temperature of primer. Again I do not think there would be any problem or any degredation problem during shipment process in the primers: because they are already validated in a handful papers and we are just following the protocol that generated during their study. Actually I am little confused about what to change.

It won't be degradation. Bright unwanted bands sound like something major is wrong somewhere. Don't always believe what you read. Methods in papers might miss crucial little details (or dare I say bullshit a bit) it sounds like you need to blast the primers for specificity and to make sure the sequence is correct. Check the sequence in the paper and the sequence you ordered, a one base typo error can make a big difference. Are you looking at variable repeat regions that can vary, this can give unwanted bands - they are common in the viruses I have worked on. Don't use touchdown. Set up separate reactions using annealing Temps 56, 58, and 60. Again predicted primer Tm can be inaccurate. Try using the extensor mastermix from thermo Scientific,t works a treat, the best I've used for all kinds of pcr assays

Good luck

Regarding the primer dimer, we have to remember that it is formed in every single PCR reaction that has been run todays. In many cases, we do not see it, but it does not mean it is not there (in reaction). PD is always formed and amplified. Most commonly it becomes a problem when we are detecting or amplifying limited amounts of target DNA load. PD is the shortest amplicon in PCR, and it has the highest yield of amplification. If the original template load is very low (

What is the template you try to amplify? Is it of the same type as in the original study you refer to?

And is the PCR mixture also exactly the same? The [MgCl2] for example, has a huge influence on the Tm of the primers.

I know from experience with qPCR that samples with less template have more primer dimers. When primer dimers are formed, and you make serial dilutions of this template, the more diluted the template is, the higher the primer dimer peak in the dissociation curve analysis.

I agree that primer design algorithms cannot fully predict the behaviour of the primers, also because the starting conditions are not always entered/known exactly.

A lot of primer design algorithms have an option for a 3'GC-clamp which of course give primers with a high 3'end Tm. Although I sometimes doubt if a few complementary bases could really anneal when the annealing temperature is >50 degrees higher then the Tm of the complementary part.

Just to add to the discussion. From my own experience, accumulating the data obtained on various and different targets/experiments, I conclude that magnesium concentration is one of the factors that can affect PD formation accelerating the amplification/detection problem. The rule is that - the lower magnesium concentration, the lesser the problem with PD formation and other kind of misamplification. However, reducing the Mg2+ concentration requires to increase the cycle time that is another problem for us in Industry where we want to amplify and detect as fast as possible. For example, when I used 2 mM MgCl2 in many of my published projects, I was able to detect pretty low target loads (up to ~100 copies) using FRET probes without seeing the effect of PD on the fluorescent curve shape. Later in time, when I was using 5 mM MgCl2 vertually on the same systems I got serious problem of false-positive detection with very low Ct. PD effect on the curve is nothing compare with such a problem, at least for us in industry.

Dear Marco,

Magnesium concentration within 2-5 mM has an effect on primer Tm, but I would not call it 'huge', rather 'modest'. It has also effect on speed of polymerization as you need to increase the time of annealing/extension in PCR.

As I said earlier, PD is the shortest and the most efficiently amplified template in PCR. It tends to overrun the target template during PCR and that is why the more diluted target shows more PD on the gel. Everything is logical.

Replying to Marco Kelders comment about primer-dimer formation and contribution to this of even very short complementarity at the primers 3'-ends. In one of my experiments I used two primers having 4 GC bp 3'-complementarity. They gave me Ct point at 11-12 cycles. He is right saying that short 4 GC bp duplex do not efficiently forms at PCR temperatures >60 oC. However, few molecules of this complex still have a good chanse to form in presence of millions/billions/or trillions(I have no calculator in my hands now to be exact) molecules of the 3'-duplexed primer. These few initiate the PD formation and that brings us a problem detecting the limited target loads. Due to extremely good (commonly 100%) efficiency one molecule of PD can overrun in PCR 10-100 molecules of the initial target loads, creating 'headache' for us in industry.