Hi Max,

I am not sure if my answer will be of any help to you or not, but we usually avoid primers with possibility of mispriming non-target templates, especially at the 3' ends. We try to pick primers with exact match at at least 10 bases from the 3' end. further we try to keep at least 3 Gs or Cs within that sequence, with one at the extreme 3' end. This strategy works quite well with our low target viral assays. 

Now coming to the primer you have designed, I believe that it is not going to give you much trouble. in general, real time PCR are designed to have common annealing & extension cycles at around 60 degrees, I expect that, at such stringent thermal conditions, odds of mispriming of your overlap primers becomes significantly less.

best wishes,

SD

To avoid mispriming, primers should not be very “sticky” on their 3’ ends. A "sticky" 3' end would be one with a high G/C content (high Tm). Either the middle of the primer or the 5’ end should be "stickier". The display in Oligo makes it easy to pick primers with good specificity, by using the “Internal Stability” window at the bottom of the screen or the Analyze>Internal Stability command (both are shown below). For

example, the following primer (5’-CAGTAACAGA TACGGGCA) would show poor

specificity since its 3’ end is GC rich relative to the rest of the primer. NOTE that

the issue is the 3’ end region, not just the 3’ base!!

GC clamp

-Refers to the presence of G or C within the last 4 bases from the 3’ end of primers

-Essential for preventing mis-priming and enhancing specific primer-template binding

Avoid >3 G’s or C’s near the 3’ end

Max 3’end stability

-Refers to the maximum ΔG of the 5 bases from the 3’end of primers.

-While higher 3’end stability improves priming efficiency, too higher stability could negatively affect specificity because of 3’-terminal partial hybridization induced non-specific extension.

-Avoid ΔG < -9.

-Avoid long runs of a single base (more than three) as this can cause primer slippage and contribute to mispriming.

-Primer concentrations that are too high increase the chance of mispriming, which may result in nonspecific amplification. Primer concentrations that are limiting can  result in extremely inefficient amplification.

- it is important to use a short annealing time of 5-15 sec.  Excessively long annealing times may lead to mispriming ­induced nonspecific amplification. 

3' end

- Having four G and/or C bases at the 3' end might be useful if the primer length is short (the bases provide "a clamping effect"), especially for universal primers, which are typically used for amplifying all cDNA or gDNA in a sample. However, adding these bases may increase nonspecific priming events for gene ­specific primers.

Hi Max,

I think the primer you have designed would be OK if you run it with an annealing temperature about 60, and will not cause any problem in term mispriming. However, I would suggest you to have one or two mismatched base at the 3' end to completely avoid mispriming.

 Generally in our field, we deal with copy number variable (CNV) region and the basic rule is one or both of your primers should have a mismatched base(s) with the similar sequences that you want to avoid amplifying them. I have designed a paralogue ratio test (PRT) in which the reverse primer completely matches the undesired sequence except for few bases (between 2 to 4 bases) which, discriminate against the undesired sequence. 

Best wishes

Nzar