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gene-specific qPCR primers for a multigene family - primer design (Jan/29/2009 )

Hi all,

I am trying to design primers to analyze expression of one particular gene from a large gene family in a polyploid grass. I have cloned and sequenced 5 alleles/family members of the gene and aligned the 3' ends, including the untranslated region. The sequences have differences, but not very many and they are dispersed, i.e., a nucleotide here and there. So I am trying to figure out how to design qPCR primers to pick up the specific gene I want without amplifying other family members. I am not sure if I have enough differences in sequence to do this. I have been told that if I put the differences at the 3' end of the primer they will be more specific, but how many difference do I need? Does there have to be more than one polymorphism in a row or can they be interspersed within the primer?



In short, the more mismatches in the 3' end the better, but try and get two right next to each other close to, or on the 3' end. Having two mismatches next to each other seems to amplify the destabilising effect, past what each individual mismatch could contribute. Having this in both primers is optimal, but in many cases you can get by with having just one highly mismatching primer. An good article that might explain things further is : "Whiley DM, Sloots TP. Sequence variation in primer targets affects the accuracy of viral quantitative PCR. J Clin Virol. 2005 Oct;34(2):104-7".

Another more expensive option is to incorporate LNA's into your primers, which will allow you to design shorter primers. The reduced length of the primer, and mismatches, especially at or next to the LNA residue at the 3' end will induce a heightened destabilisation effect, thus making the primer more specific to its homologous sequence.


You have a good chance of preventing a product with a single 3' mismatch. It would help if the mismatch is purine:purine, but most natural mutations will not be of this form.


grassgirl on Jan 29 2009, 11:04 AM said:

how many difference do I need? Does there have to be more than one polymorphism in a row or can they be interspersed within the primer?

So, people do design primers for SNP analysis, which is a SINGLE nucleotide. So, does it have to be more than one, no. Will it help if you have more than one? Probably yes. I'd look for articles about designing primers for SNP (or "snip") analysis. And yes, the 3' location of the polymorph in the primer supposedly will help with detection specificity.


Thanks to all who replied. Very useful information.


Try having as meny mismatches as possible at the 3' end of your primers.
If the 3' end doesn’t mach up it won't elongate!!!!
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