dG values in primer design are very important in order to prevent homodimerisation primer dimers etc.
A critical step in primer design is the in silico analysis of your primer pairs and amplicon.
A number of programs can therefore be used (eg mFOLD). On several fora there is some debate about the acceptable dG values primers may have in order to be acceptable.
Is there anybody who knows or did real wetlab studies in order to determine the accepatable cutoff of theoretically found dGs?
Some say that dG = -10 kcal/mol is still acceptable
Others say a dG of =-2 kcal/mol is still acceptable
Thanks in advance for any answer.
Hi Stefan!
I agree with all previous answers made by colleagues above and feeling compelled contributing my 10 cents to you as well. As scientists we never know how difficult it will be the next sequence we will be working with and how difficult primer design will turn because it will depend on its putative % of GC content etc... so narrowing the delta G values may turn primer design fustrating
Making reference to mFOLD, we assume primer sequences between -3 to +3 Kcal/mol (in plot) good, Usually favorable to amplify your target, however, Delta G values + or - around 0 are better, lets say -1 to +1 may be optimal. Free energy at the 'spot' should be preferable negative . Lets say 0 to -3. Alternatively if using Primer3, primers with ANY scores between 0 and 3 are optimal if keeping the 3' terminus without secondary structure (0). Still... if you are not able to get a good primer because the thermodymanics of the sequence it self (GC reach or so) you can design 5' A/T non complementary extensions or flaps to improve your primer performance (see Mol Biotechnol (2013) 55:17–26 and/or DOI 10.1007/s12033-012-9617-5 ). Hope this help.
I work on Patented FRET Primer-primer, Primer-probe and FRET quenching in distant primer-primer model systems. In my 3 and half years of experience of designing and using such oligos in qRT-PCR i have restricted myself to atmost 3 dA-dT dimers and 1 dC-dG dimer at the 3'-end. I would not use an oligo dimered at 3'-end which account for lower than -6kcal/mole. I would consider atmost 6bps of non-terminal dimers between longmers (>22bases) which could account for upto -11kcal/mole. In case of 5'-end, to avoid too much anchoring, i would prefer AT-rich dimers of atmost 5bps which account up to -9kcal/mole. 5'-end dimers does good in reducing non-specific amplicon build-up. This criteria holds good if annealing temperature is used at or above 60c. In case of annealing temperatures
Hi,
I work with a company that performe my PCR produts' sequencing. The company recommend dG higher than -1 kCal/mol, to ensure that the primer will not forme self annealing.
I hope it helps.
Ana
I could be difficult to acheive ~1kcal/mole in most of the cases. If achevied then pcr efficiency should be at its best.
Hi Stefan!
I agree with all previous answers made by colleagues above and feeling compelled contributing my 10 cents to you as well. As scientists we never know how difficult it will be the next sequence we will be working with and how difficult primer design will turn because it will depend on its putative % of GC content etc... so narrowing the delta G values may turn primer design fustrating
Making reference to mFOLD, we assume primer sequences between -3 to +3 Kcal/mol (in plot) good, Usually favorable to amplify your target, however, Delta G values + or - around 0 are better, lets say -1 to +1 may be optimal. Free energy at the 'spot' should be preferable negative . Lets say 0 to -3. Alternatively if using Primer3, primers with ANY scores between 0 and 3 are optimal if keeping the 3' terminus without secondary structure (0). Still... if you are not able to get a good primer because the thermodymanics of the sequence it self (GC reach or so) you can design 5' A/T non complementary extensions or flaps to improve your primer performance (see Mol Biotechnol (2013) 55:17–26 and/or DOI 10.1007/s12033-012-9617-5 ). Hope this help.
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