PCR Primer Design
Poor primer PCR design is the one major cause of all failures or false positives or negatives in single and multiplex PCR. Primers may fail for many different reasons and the major factors that affect PCR primer designs are:
- Assay temperature which affects the equilibrium constant of oligo binding, hence the amount of primer that binds its target.
- Cation concentration (K+, Na+, Mg2+, NH4+) which affects the stability of one oligonucleotide binding to another.
- Buffer additives / consistencies such as denaturants or viscosity. Current buffer additives include glycerol, DMSO, formamide, TMAC and betaine, all of which will affect the thermodynamic stability of oligo hybridization.
- Desired annealing temperatures for primers and probes, which is related to oligo length and amplicon size.
- Secondary structure which affects any oligonucleotide and its ability to bind to another oligonucleotide or itself. Visual OMP uses a dynamic programming algorithm to calculate the energy of all possible structures by using our thermodynamic database. The optimal and nearby suboptimal structures are predicted to be the most populated at equilibrium.
- Synthetic nucleotide modification, such as modified backbones, sugars or bases that have the ability to increase or decrease target binding.