Sophisticated algorithms for qPCR success
Eurofins Genomics' qPCR Assay Design Tools are based on Prime+ of the GCG Wisconsin Package enhanced with additional parameters for perfect probe design.
In selecting appropriate primer/probe combinations for genotyping and gene expression assays, a variety of constraints on the primer, probe and amplified product are considered and partly predefined as default values in our qPCR assay design:
- qPCR probe criterias
Optimimum properties for uniqueness, annealing and self-annealing (probe length, GC) are used. Sequence homopolymer stretches and 5' G are avoided and more Cs than Gs are present in the probe sequence. In addition probes are designed with a Tm 8-10 °C higher than the Ta of the PCR product.
- Primer criterias
Optimum primer lengths, GC-content and primer Tm are used. Optimum Tm difference between both primers are considered and G/C as primer 3' clamp is recommended. For efficient priming, primers with extensive self-dimer and cross-dimer formations are avoided to minimise primer dimer formations and secondary structures.
- Criterias of the amplicon
Optimimum amplicon size, GC-content and Tm are suggested as default values.
For the biplex qPCR assay design all primers and probes are cross-checked against each other.
The results are scored according to the best predicted performance criteria based on the lowest annealing score of the probes.
Selected primer / probe combination can directly be ordered online.