Is a 3´ end heterodimer of Delta G: -1.6 kcal/mole with 2 base pairs of bridge going to affect the real-time PCR primer efficiency?
I would like to know of someone who has used primers with these conditions and has got a good efficiency.
I think this may not have much effect on efficiency unless if you set the Annealing temperature of primers is equal or lesser than the hair pin melting temperature during amplification. For calculating the melting temperature of hairpin you can follow this link:
http://eu.idtdna.com/calc/analyzer
3´ end heterodimer of Delta G: -1.6 kcal/mole with 2 base pairs?
The question is if it influences efficieny. The answer is not simple.
First try to avoid t3' complementarity and use commercial software such as Allele ID or beacon designer.These programs rank primers/probes with respect to the chance of correct products. This chance refers to much more than 3' complentarity. In my experience Primer 3 and primer blast are inferior,! Check during optimization phase amplification by melting curve analysis. Check qPCR's aimed at probe assays always with intercalting dyes in the first experiments!!! Non- specific products lower the efficiency of the synthesis of your specific amplification by using dNTP's, occupation of enzymes and use of primers.
In general: The effect of 3' complmentarity depends on a) which platform (probe, intercalating dye) b) optimization of the PCR, c) initial concentration of targets. The lower target concentration, the more chance of dimers and other non- specific products.. When using intercalating dyes, you cannot discriminate between specific and non- specific products and sensitivity will be overestimated
Hairpins: -1
Self dimer: -1.6 kcal /mole in 3´
cross dimer: all secondary structures > -6kcal/mole with no inconvenients there
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