A significant aspect of PCR primer design is to ensure a sufficient primer annealing efficiency. The annealing efficiency at annealing step can determine PCR yield.
- We have developed primer/amplicon design tools based on rigorous chemical equilibrium equations and thermodynamic calculations which are capable of producing quantitative predictions of primer annealing efficiency. Our design ensures the annealing efficiency of each primer to reach a threshold level (e.g. >0.95) at a predetermined annealing temperature.
- As comparison, current PCR primer designs are generally based on melting temperature (Tm) calculation. The method is largely empirical and does not provide quantitative assessment of primer-template interactions at various PCR stages. For example, by knowing the Tm alone of a primer sequence one may not know exactly what annealing efficiency will be at a predetermined annealing temperature.
DNA sequence Tm is defined as the temperature of an equilibrium system in which 50% of the complementary sequence pairs remain associated to each other as double strand sequences.
10 primer sequences are selected from human PIK3CA12, LMTK2, LTK, TP53 genes with a narrow Tm range of 69.6 to 70.3°C.
fa varies from 0.891 to 1.000 at an annealing temperature of 65°C. After 20 PCR cycles, the contributions to the final product yields from the primers would vary from 0.89120 = 0.099 to 1, which is more than 10-fold variation.
PCR buffer composition is cPrm=500 nM, cTpl=1 fM, Tanneal=65°C, cNa=0.06M, and cMg=0.0015M. GC contents of the sequences are between 0.2 and 0.8.