Intro & Info
Eurofins Genomics' qPCR Assay Design Tools are based on Prime+ of the GCG Wisconsin Package enhanced with additional parameters for perfect probe design
The qPCR Assay Design tool analyses the entered DNA sequence and chooses the optimum qPCR Probes and PCR primer pairs. In selecting the appropriate probe and primers, a variety of constraints on the probe, the primers and amplified product are already considered and taken as default values. You either can use the default constraint values or modify those values to customise the analysis.
Copy & paste the target sequence from an external source. DNA sequence information as well as FASTA sequences ( starting with an ">" followed by the name) are possible. Line breaks and blank spaces are allowed. The template sequence may contain ambiguous bases, but the design tool will not select primers or probes complementary to any ambiguous sites on the template sequence.
You can design primers and probes from the whole template (= target sequence) or limit the choices to a particular region. For PCR primer pairs, you can specify any required bases at the 3' end of the primer (3' clamp), and a maximum difference in primer melting temperatures.
qPCR Probe Criterias
For the qPCR probe you can specify minimum and maximum probe length and GC content of the probe.Sequence homopolymer stretches and 5' G are avoided by the software automatically and more Cs than Gs are considered to be present in the probe sequence. In addition probes are designed with a Tm 8-10 °C higher than the Ta of the PCR product.
For the PCR primer pairs you can specify minimum and maximum primer length, primer GC content and primer melting temperature (Tm). The maximum primer length you can search for are 100 bases. Primer melting temperatures is calculated by using the nearest-neighbor model of Borer, and thermodynamic parameters for DNA nearest-neighbor interactions and the salt dependence of oligonucleotides determined by SantaLucia [Proc. Natl. Acad. Sei. USA. 95; 1460-1465 (1986)]. For efficient priming, the design tool avoids primers with extensive self-dimer and cross dimer formations in order to minimize primer secondary structure and primer dimer formation.
For PCR products, you can specify a range of acceptable product sizes and define the minum and maximum GC content and melting temperature (Tm). The maximum product length to enter is 5,000 bases. PCR product melting temperatures are calculated by using the formula of Baldino, et al. [in Methods Enzymol. 168; 761-777 (1989)] as modified slightly by Rychlik, et al. [Nucleic Acids Res. 18; 6409-6412 (1990)].
Salt concentration specifies the mM salt concentration in the reaction. This value is used in the calculation of both primer and PCR product melting temperatures. The default value is 50.0 and the value may range from 0.1 to 50.0.
Primer concentration specifies the nM concentration of primer DNA in the reaction. This value is used in the calculation of primer melting temperature. The default value is 50.0 and the value may range from 0.1 to 50.0.
Click on the "Design qPCR Assay" button to get a list of optimum probe / primer combinations according to your output parameter settings.