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Interpreting primer BLAST scores for self-complementary


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#1 bjk1985

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Posted 18 July 2014 - 11:25 AM

I can't find this info anywhere. I designed a primer pair w/IDT tools and they came out fine for homo- and heterodimers along with hairpin formation. Then when I went to do a primer pair BLAST to make sure that I got things right w/repsect to orientation... it tells me that I have "self complementarity" scores of 7 and 6 (F and R, repsectively) and "self 3' complementarity" scores of 4 and 6. I attached the relevant image. Given that IDT told me I was fine, is this a concern (again, I can't find anything explaining these scores in terms of when to be concerned)?

Attached Thumbnails

  • primers for BMV.jpg

Edited by bjk1985, 18 July 2014 - 11:27 AM.


#2 bjk1985

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Posted 29 July 2014 - 02:29 PM

Just to update for information purposes in case anyone else has the same question sometime and gets to this thread by using the search feature...

 

The primers worked.



#3 Trof

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Posted 02 August 2014 - 01:22 AM

Both of the calculations are just approximate I would say. I don't know how is either of them calculated in detail, co it's hard to tell if one of those is better.

 

But, have in mind, that usualy both have several parameters like salt and oligo concentration, that you can set. Maybe they use different settings or maybe they don't use some of the concentrations at all. It would be logical to expect that different enviroment of oligos have different effects on their self-alignment.

 

Also some of the polymerase mixes may have more problems with dimers than others (depending on the enviroment thing again, but it means that using some polymerase, primers will work better than in other). For example hotstart PCR prevents unspecific amplification in general, ale dimers are unspecific in their nature (the complementarity is not 100%). The higher specificty, the less dimers.

 

But, I suppose in any case, primers with high self-binding will "work" most of the time. The self-binding tells you how much they like themselves instead of the template. In different conditions again, the dimers will form more. For example, I thing using too high primer concentration:template ratio, will lead to preferential formation of dimers (since there is overabundance of primers), and dimers will form more likely, and when they do, they use up the polymerase and nucleotides, so less of it is available.

 

But to sum it up, all this thought excercise, when you design in IDT and it tells you it's fine (but in my opinion, IDT images of dimers is usually pretty confusing, you must know what deltaG is fine and which not, using primer3 (which is the algorithm in PrimerBLAST) is simplier, that it only tells you number, you know that high is bad, and try to avoid it) it's probably going to work. If you design primers in primer3 and use their recommended setting, it's probably also going to work.

Regardless on what different algorithms say.

 

And just to make it more complicated. I personaly seen primers, that have so bad in silico parameters that I though they never gonna work. And they did. Because all these tools will never tell you which primers will work, but they will try their best to point out which has higher probability to not work. And they still may, specific sequence, specific conditions.. these ale all unpredictible. 

 

So try to design "good" primers, but it's not a rule that "bad" will never work (or that "good" ones will). Just probability.


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#4 bjk1985

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Posted 31 August 2014 - 09:08 AM

I thought the IDT software said, with respect to the likelihood of inter- and intraprimer binding, that anything larger than a delta G of -9 (so any value 0 to -8.9) is supposed to be ok as a general rule of thumb.



#5 phage434

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Posted 31 August 2014 - 10:52 AM

The major failing of the IDT tools is that they fail to distinguish homo or heterodimer binding which allows extension of the primers in the 3' direction (fatal) from those that bind in much more benign ways.

I don't know if the primer3 tools do this or not.



#6 Trof

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Posted 17 September 2014 - 12:50 AM

Yes, primer3 has different scores for stability anywhere in the sequence and for 3' end stability, which is kept low as possible. This is calculated for both single primer and for primer pair.

It has no fancy pictures, but you can set your own treshold on everything.


Our country has a serious deficiency in lighthouses. I assume the main reason is that we have no sea.

I never trust anything that can't be doubted.

'Normal' is a dryer setting. - Elizabeth Moon





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