Università degli Studi di Salerno
Question
Asked 29th Feb, 2012
dG value of Primers
This a self annealing result of an upper primer from OLIGO EXPLORER
5'-CATCGCACGTCGTCTTTC-3'
: : : | | |
3'-CTTTCTGCTGCACGCTAC-5'
dG: -1.74 kcal/mol
What does the dG value denote?
And what range of dG values are acceptable when designing primers?
Most recent answer
There are some general guidelines.
Your primers are perfect until they work.
Check always the sequence to be amplified.
Tehere--> Stability is a consequence.
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Popular answers (1)
Temuco Catholic University
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.
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All Answers (17)
Temuco Catholic University
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.
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Institute of Genomics and Integrative Biology
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.
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City College of New York
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.
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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)
5 Recommendations
University of Groningen
@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.
1 Recommendation
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.
National Institute of Technology, Arunachal Pradesh
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 .
Università degli Studi di Salerno
Send me the sequence.
G and C increases primer dimers formation.
Theodore - it's correct if sequence will let you.
University of Groningen
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 :)
Shahid Chamran University of Ahvaz
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
1 Recommendation
The dG value varies with temperature. Some software calculate dG at 25 Celsius, some at 37 Celsius, more intelligent software let you set the temperature. In the first approximation, dG = dH - TdS. I would compare dG values for your structures at the temperature of your experiment (Texp), e.g. annealing temperature (Texp=60 Celsius). <b>dG at much lower temperature is not that useful for design of your experiment.</b> Simple rules like dG larger or smaller by 3 kcal/mol are too crude approximations to be much useful. I would hesitate to use them.
If the folded DNA structure is not desired, its dG is ideally positive at Texp and the structure will not form. If several folded structures are possible (dG < 0), the structure with the lowest dG will most likely be most prevalent. From dG, you can calculate equilibrium constants between folded and unfolded DNA, dG = -RTln Ka. From Ka, you can calculate the concentration of each folded structure in solution. That is the key info. The enzyme reaction depends on concentrations of reactants. This calculation is not that simple math if many folded structures are possible..
---------------------------------------------------------
Since Ka is exponential function of dG, Ka = exp (-dG/RT), tiny changes in dG have huge impact on Ka and amounts of the folded structure.
---------------------------------------------------------
I would compare dG values with dG of completely folded duplex at the temperature of experiment. If the dG differ by more than 25%, the folded structure with lower dG will be in large excess and you do not have to worry about the structure with less negative dG.
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Centre Hospitalier Universitaire de Nice
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
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