MFEprimer: Multiple factor evaluation of the specificity of PCR primers

Enter primer sequences (Demo primers)

For a pair of primers	For multiple primers (Fasta format required)
Forward primer:
Reverse primer:
	Or, upload FASTA file:

Select database OR upload my custom database in FASTA format

Or, upload my custom database:

Example

If the databases did not meet your demand, you can upload your custom database to our server in FASTA format. For example, if you want to check the specificity of your primer set(s) for viral or bacterial genome, you can save the target sequences as "PCR templates" to a file in FASTA format and upload it. Here is an example of the custom database. The size of your custom database should be less than 10 Mb. Otherwise, we strongly recommend you to use the (stand-alone version of MFEprimer).

General settings

Word size for BLASTN	E-value for BLASTN	Amplicon size to display	PPC cutoff value (%)
		Between and

Inherently, MFEprimer can predict all of the possible amplicons. The sensitivity of MFEprimer depends on the parameters such as E-value, Word size. The user can define an expected amplicons size range to view the amplicons.

PPC (Primer Pair Coverage) was defined to score the ability of the primer pair (a forward primer coupled with a reverse primer) binding to the DNA template. Click here for details.

E-value is a parameter used in NCBI BLAST (Altschul SF et al. J Mol Biol. 1990 Oct 5;215(3):403-10). MFEprimer uses NCBI BLAST to check the sequence similarity between primers and the DNA templates. By using a larger E-value, BLAST would report more hits. As a result, MFEprimer would be more sensitive.

Word size is a parameter used in NCBI BLAST (Altschul SF et al. J Mol Biol. 1990 Oct 5;215(3):403-10). MFEprimer uses BLAST to check the sequence similarity between primers and the DNA templates. With smaller word size, BLAST would report more hits. As a result, MFEprimer would be more sensitive.

Advanced settings

These settings are used for calculating the Gibbs free energy and melting temperature. The default settings are: concentration of monovalent cations: 50.0 mM; concentration of divalent cations: 1.5 mM; annealing oligo concentration: 50.0 nM; concentration of dNTPs: 0.25 mM. These are standard PCR conditions (see http://www.k-state.edu/hermanlab/protocols/StandardPCRConditions.html). See FAQ for details.

Concentration of monovalent cations (usually KCl, mM)	Concentration of divalent cations (usually MgCl₂, mM)	Concentration of dNTPs (mM)	Annealing oligo concentration (nM)

The millimolar concentration of salt (usually KCl) in the PCR reaction. MFEprimer uses this argument to calculate primer melting temperatures.

The millimolar concentration of divalent salt cations (usually MgCl₂) in the PCR reaction. MFEprimer converts concentration of divalent cations to concentration of monovalent cations using formula (7) proposed in the paper Ahsen et al., Clin Chem. 2001 Nov;47(11):1956-61..

The millimolar concentration of deoxyribonucleotide triphosphate. This argument is considered only if concentration of divalent cations is specified.

The nanomolar concentration of annealing oligos in the PCR reaction. MFEprimer uses this argument to calculate primer melting temperature. The default (50 nM) works well with the standard protocol used at the Whitehead/MIT Center for Genome Research--0.5 microliters of 20 micromolar concentration for each primer oligo in a 20 microliter reaction with 10 nanograms template, 0.025 units/microliter Taq polymerase in 0.1 mM each dNTP, 1.5mM MgCl₂, 50mM KCl, 10mM Tris-HCL (pH 9.3) using 35 cycles with an annealing temperature of 56 degrees Celsius. This parameter corresponds to 'c' in Rychlik, Spencer and Rhoads' equation (ii) (Nucleic Acids Research, vol 18, num 21) where a suitable value (for a lower initial concentration of template) is "empirically determined". The value of this parameter is less than the actual concentration of oligos in the reaction because it is the concentration of annealing oligos, which in turn depends on the amount of template (including PCR product) in a given cycle. This concentration increases a great deal during a PCR; fortunately PCR seems quite robust for a variety of oligo melting temperatures.