Because it took me awhile to locate the answer, I'll share it here. The scores are output from PRIMER3 algorithm (which powers the NCBI's PrimerBLAST website as well) and is described in great detail at http://primer3.sourceforge.net/primer3_manual.htm#PRIMER_MAX_SELF_ANY

In short, the maximum theoretical self complementarity score (globally or only at 3' end) is the length of the primer itself. The 3'-self complementarity is useful to predicting primer-dimers, while the self-complementarity score is useful to predict possible secondary structures between the primer itself or even primer pairs. As described by PRIMER3 manual, there is an option to specify the maximum tolerated self complemntarity score: PRIMER_MAX_SELF_ANY. The default value is 8.00.

I don't believe there is any estimated cut-off or "safe" value for these "scores" as they are more qualitative than quantitative. Lower is better, and the primers suggested by PRIMER3 usually have similar values. I have never systematically tried several different primer combinations to see if these scores ultimately predict well problems with PCR. I have used one of the top 10 suggested (default list shows up to 10 I think) many times and they have worked often, and when they don't it's more often a problem with my PCR mix or template rather than primer sequence. That said, I have yet to see primers suggested by PRIMER3 or NCBI's PrimerBLAST to have values exceeding 7-8 from a typical 20mer primer (about 33%). so I would be cautious with a sequence having a score >50%.

If youre thinking of using the primers for SYBR based qPCR assay then avoid all dimers formation as much as possible, especially on the 3' site.

If thats not possible then ΔG of about 2 should be acceptable but dimers formation on the 3' site should be avoided at all costs.

Probe based qPCR assay can be less restrictive on these scores

Specificity of the assay can only be confirmed/validated after experimentation

Thanks Raditijo.

We will use a SYBR based assay.

Self complementarity ranges from 2 to 6, and self 3' complementarity from 0 to 3 in the different primer pairs.Are these reasonable values? Do they correspond to the deltaG values you mention?

We used the PrimerBlast designer from GenBank. Would you recommend any particular primer design package? I know there are many - it's a difficult choice.

I never use PrimerBlast from GenBank so I dont know if ΔG values correspond to that of PrimerBlast but most primer design programs use them.

I usually use the free browser software Beacon Designer to quickly check for dimers formation between primers and probes if you have  one. Check out the link below.

I have to correct myself regarding ΔG values, since negative values means the dimers are more stable than dimers with positive ΔG values. so -6 ΔG means dimers more stable than the one with 2 ΔG value

http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1

Hi John,

I remember using a free tool from operon (www.operon.com) for checking potential primer-dimer formation. They also have a free online tool for designing primers, which might be work checking. 

Thermo also has a free online check :

http://www.thermoscientificbio.com/webtools/multipleprimer/

Saying that, primers pairs are quite cheap and the only way to be sure on how good are they is trying them. A single and sharp peak in the melting curve analysis is a good news, that can be further verified by cloning and sequencing of the products. I am happy to help, if needed..

All the best, 

Hi Mauricio, thanks for the tip. I've checked all the tools mentioned: the Beacon designer Raditijo mentioned seems to give more information. I need to try a few more combinations though.

Thanks guys.

I had a scout around and found this very useful and detailed explanation of most parameters used to describe primers, particularly the delta G (Gibbs free energy) issue:

http://www.premierbiosoft.com/tech_notes/PCR_Primer_Design.html

Because it took me awhile to locate the answer, I'll share it here. The scores are output from PRIMER3 algorithm (which powers the NCBI's PrimerBLAST website as well) and is described in great detail at http://primer3.sourceforge.net/primer3_manual.htm#PRIMER_MAX_SELF_ANY

In short, the maximum theoretical self complementarity score (globally or only at 3' end) is the length of the primer itself. The 3'-self complementarity is useful to predicting primer-dimers, while the self-complementarity score is useful to predict possible secondary structures between the primer itself or even primer pairs. As described by PRIMER3 manual, there is an option to specify the maximum tolerated self complemntarity score: PRIMER_MAX_SELF_ANY. The default value is 8.00.

I don't believe there is any estimated cut-off or "safe" value for these "scores" as they are more qualitative than quantitative. Lower is better, and the primers suggested by PRIMER3 usually have similar values. I have never systematically tried several different primer combinations to see if these scores ultimately predict well problems with PCR. I have used one of the top 10 suggested (default list shows up to 10 I think) many times and they have worked often, and when they don't it's more often a problem with my PCR mix or template rather than primer sequence. That said, I have yet to see primers suggested by PRIMER3 or NCBI's PrimerBLAST to have values exceeding 7-8 from a typical 20mer primer (about 33%). so I would be cautious with a sequence having a score >50%.

My question is the exact same. I just want to understand the scores. I am using the NCBI primer-blast website and want to know if the self complementarity score at value 4.00 is alright? The self 3' complementarity is 0.00 for both my forward and reverse primers. I am also doing a qPCR (for plant gene expression).

The detailed description of how the score is calculated is given in http://primer3.sourceforge.net/primer3_manual.htm#PRIMER_MAX_SELF_ANY

I'll copy it here for quick reference:

PRIMER_MAX_SELF_ANY (decimal, 9999.99; default 8.00)

PRIMER_MAX_SELF_ANY describes the tendency of a primer to bind to itself (interfering with target sequence binding). It will score ANY binding occurring within the entire primer sequence.

It is the maximum allowable local alignment score when testing a single primer for (local) self-complementarity and the maximum allowable local alignment score when testing for complementarity between left and right primers. Local self-complementarity is taken to predict the tendency of primers to anneal to each other without necessarily causing self-priming in the PCR. The scoring system gives 1.00 for complementary bases, -0.25 for a match of any base (or N) with an N, -1.00 for a mismatch, and -2.00 for a gap. Only single-base-pair gaps are allowed. For example, the alignment

5' ATCGNA 3'

|| | |

3' TA-CGT 5'

is allowed (and yields a score of 1.75), but the alignment 5' ATCCGNA 3'

|| | |

3' TA--CGT 5'

is not considered. Scores are non-negative, and a score of 0.00 indicates that there is no reasonable local alignment between two oligos.

http://primer3.sourceforge.net/primer3_manual.htm#PRIMER_MAX_SELF_ANY

The lower is the better

The lower the score, the lower self complementarity.

Its clear that the lower the better, but how low is good enough, I think this was the original question, but no straight answer as far as I understood.

My lab has used primers with high complementary scores, and received great results. And alternatively used primers with low complementary scores, yet received poor results... Sometimes the best way to check things... is to just run a sample to see how well the melt curve and product amplification curve turns out...

Interactions seen in the real world cannot always be taken into account by these programs.

Don't over-think things.

Best of luck,

Colin

so helpful