In simply terms, dG represents the quantity of energy needed to fully break a secondary DNA structure. The lower dG values (more negative values), the higher the quantity of energy needed to "separate" the DNA strands if a secondary structure (a primer dimer or hairpin, for instance) is formed. This is very important in PCR reaction because in that cases, the energy is given for temperature. If your primers have very negative values of dG, the quantity of energy needed to break the dimer is higher, or what is the same, you need higher temperatures, wich can disturbe the reaction. In general terms, values of dG more positives than - 9 kcal/mol are acceptable. In my experience, values up to -6 kcal/mol for internal dimmers and -5 for 3' end primers give me good results. In your case, the structure formed is irrelevant, first, because the dG value is acceptable low, and second, the bases that bind in this structure aren't in 3' end of primer, therefore, no amplification of this structure should occur.

In simply terms, dG represents the quantity of energy needed to fully break a secondary DNA structure. The lower dG values (more negative values), the higher the quantity of energy needed to "separate" the DNA strands if a secondary structure (a primer dimer or hairpin, for instance) is formed. This is very important in PCR reaction because in that cases, the energy is given for temperature. If your primers have very negative values of dG, the quantity of energy needed to break the dimer is higher, or what is the same, you need higher temperatures, wich can disturbe the reaction. In general terms, values of dG more positives than - 9 kcal/mol are acceptable. In my experience, values up to -6 kcal/mol for internal dimmers and -5 for 3' end primers give me good results. In your case, the structure formed is irrelevant, first, because the dG value is acceptable low, and second, the bases that bind in this structure aren't in 3' end of primer, therefore, no amplification of this structure should occur.

Thanks mate : )

In practice, enthalpy (dH°) and free energy (dG°) values for each of the 10 possible Watson-Crick

DNA pairwise interactions are used to calculate pairwise entropy (dS°) values via the following

standard equation:

dG° = dH° - TdS°

Note: T is temperature in °K.

Interaction dH° kcal/mol dS° cal/mol per °K dG° kcal/mol

AA/TT -9.1 -24.0 -1.9

AT/TA -8.6 -23.9 -1.5

TA/AT -6.0 -16.9 -1.0

CA/GT -5.8 -12.9 -2.0

GT/CA -6.5 -17.3 -1.3

CT/GA -7.8 -20.8 -1.6

GA/CT -5.6 -13.5 -1.6

CG/GC -11.9 -27.8 -3.6

GC/CG -11.1 -26.7 -3.1

GG/CC -11.0 -26.6 -3.1

XX/XX -6.0 -16.9 -1.0

I agree with Alfonso.

Small comment here. At times acceptable range of dG values depend upon application in which primers will be used. This is especially critical for real-time PCR. The reason is that when dG is lower than certain critical value (it may or may not depend on the type of annealing: self, between primers, at 3 or 5'end) primer/primer pairs may not work efficiently or at all.

From what I have been reading the type of self annealing you are observing as well as the dG value should NOT create any problem.

Usually acceptable values for primer sequences DG are:

when you examine your primer sequences for hairpins: -2 kcal/mol (3' end)

-3 kcal/mol (internal)

when you examine your primer sequences for self/cross dimers: -5 kcal/mol (3' end)

-6 kcal/mol (internal)

I am also agree with alfanso

Avoid C and G to 5', 3' end.

GC content

@Massimiliano- Why should I avoid G or C at the 3' end? Doesn't that help my primer to bind more strongly to the template.

That's correct but you should not have more than three G or C in the last five bases of the 3' end of your primer.

dg value is the energy requireb to free DNA from its secondary structure to get linear or uncoiled ready for transcription and translation.

dg value of the Gc should always be lower than AT content in reverse priming and higher for forward priming

or from 3'-5' priming / 5'-3' priming.

Tm < tp .

Send me the sequence.

G and C increases primer dimers formation.

Theodore - it's correct if sequence will let you.

5'-CATCGCACGTCGTCTTTC-3'

: ||| :

3'-GCCGCGCATCTTTTTCATAA-5'

dG: -3.54 kcal/mol

5'-CATCGCACGTCGTCTTTC-3'

:: ||| :

3'-GCCGCGCATCTTTTTCATAA-5'

dG: -1.74 kcal/mol

5'-CATCGCACGTCGTCTTTC-3'

: |||

3'-GCCGCGCATCTTTTTCATAA-5'

dG: -1.74 kcal/mol

These are my primer dimers. But I don't think these have significant dG values (As per all the posts above). Anyways these primers work fine for me in PCR. Asked this doubt so that I can learn more about primer designing.

Thank you Massimiliano :)

G and C don't increase primer dimer formation, they increase stability of primer dimers structure because of formation three bound...

I agree with Nancy , dG value hes become fewer than ..... because of tdG value unit is Kcal/mol but we use primers in pmol concentration in PCR reaction

using DMSO as PCR additive decrease Primer dimer formation

Dear Colleagues,

For the less specialised in thermodynamic, but PCR user as I am, finally, the known data that it is said that for primers , you don't have to use more than a certain pourcentage of C and G , compared to A and T, correspond to all these calculations, isn't it ?

Best regards

Didier J

There are some general guidelines.

Your primers are perfect until they work.

Check always the sequence to be amplified.

Tehere--> Stability is a consequence.