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Detailed primer and probe parameters
A large number of criteria for the primer design can be set. Setting and adjustments of parameters results in dynamic updates of the primer calculations and the output of the calculations. The program thus offers full interactivity in your dynamic search for the best primers.
The following criteria can be set for single primers:
- Length. Determines the length interval within which primers can be designed by setting a maximum and a minimum length. The upper and lower lengths allowed by the program are 50 and 10 nucleotides respectively.
- GC content. Determines the interval of CG content (% C and G nucleotides in the primer) within which primers must lie by setting a maximum and a minimum GC content.
- Melting temperature. Determines the temperature interval within which primers must lie. When the Nested PCR or TaqMan reaction type is chosen, the first pair of melting temperature interval settings relate to the outer primer pair i.e. not the probe. The melting temperature group can also be unfolded to show parameters regarding the reaction mixture:
- Primer concentration. Specifies the concentration of primers and probes in units of nanomoles (nM)
- Salt concentration. Specifies the concentration of monovalent cations ([NA+], [K+] and equivalents) in units of millimoles (mM)
- Inner melting temperature. This option is only activated when the Nested PCR or TaqMan mode is selected. In Nested PCR mode, it determines the allowed melting temperature interval for the inner/nested pair of primers, and in TaqMan mode it determines the allowed temperature interval for the TaqMan probe.
- Self annealing. Determines the maximum self annealing value of all primers and probes. This determines the amount of base-pairing allowed between two copies of the same molecule. The self annealing score is measured in number of hydrogen bonds between two copies of primer molecules, with A-T base pairs contributing 2 hydrogen bonds and G-C base pairs contributing 3 hydrogen bonds.
- Self end annealing. Determines the maximum self end annealing value of all primers and probes. This determines the amount of consecutive base pairs allowed between the ends of two copies of the same molecule. This score is also calculated in units of hydrogen bonds between two primer copies of identical primer molecules.
- Secondary structure. Determines the maximum score of the optimal secondary DNA structure found for a primer or probe. Secondary structures are scored by the number of hydrogen bonds in the structure, and 2 extra hydrogen bonds are added for each stacking base-pair in the structure.
- 3' end G/C restrictions. When this checkbox is selected it is possible to specify restrictions concerning the number of G and C molecules in the 3' end of primers and probes. A low G/C content of the primer/probe 3' end increases the specificity of the reaction. A high G/C content facilitates a tight binding of the oligo to the template but also increases the possibility of mis-priming. Unfolding the preference groups yields the following options:
- End length. The number of consecutive terminal nucleotides for which to consider the C/G content
- Max no. of G/C. The maximum number of G and C nucleotides allowed within the specified length interval
- Min no. of G/C. The minimum number of G and C nucleotides required within the speci_ed length interval
- 5' end G/C restrictions. When this checkbox is selected it is possible to specify restrictions concerning the number of G and C molecules in the 5' end of primers and probes. A high G/C content facilitates a tight binding of the oligo to the template but also increases the possibility of mis-priming. Unfolding the preference groups yields the same options as described above for the 3' end.
- Mode. Specifies the reaction type for which primers are designed
- Standard PCR. Used when the objective is to design primers, or primer pairs, for PCR amplification of a single DNA fragment.
- Nested PCR. Used when the objective is to design two primer pairs for nested PCR amplification of a single DNA fragment.
- Sequencing. Used when the objective is to design primers for DNA sequencing.
- TaqMan. Used when the objective is to design a primer pair and a probe for TaqMan quantitative PCR.
