Enter the position ranges if you want the primers to be located on the specific sites. The positions refer to the base numbers on the plus strand of your template (i.e., the "From" position should always be smaller than the "To" position for a given primer). Partial ranges are allowed. For example, if you want the PCR product to be located between position 100 and position 1000 on the template, you can set forward primer "From" to 100 and reverse primer "To" to 1000 (but leave the forward primer "To" and reverse primer "From" empty). Note that the position range of forward primer may not overlap with that of reverse primer.
Optionally enter your pre-designed forward primer. Always use the actual primer sequence (i.e., 5'->3' on plus strand of the template).
Optionally enter your pre-designed reverse primer. Always use the actual primer sequence (i.e., 5'->3' on minus strand of the template).
With this option on, the program will search the primers against the selected database and determine whether a primer pair can generate a PCR product on any targets in the database based on their matches to the targets and their orientations. The program will return, if possible, only primer pairs that do not generate a valid PCR product on unintended sequences and are therefore specific to the intended template. Note that the specificity is checked not only for the forward-reverse primer pair, but also for forward-forward as well as reverse-reverse primer pairs.
Enter an organism name, taxonomy id or select from the suggestion list as you type. [?]
It is strongly recommended that you always specify the organism if you are amplifying DNA from a specific organism (because searching all organisms will be much slower and off-target priming from other organisms are irrelevant).
Genome database (reference assembly from selected organisms): Sequences from selected organisms including apis mellifera, arabidopsis, bos taurus, danio rerio, dog, drosophila melanogaster, gallus gallus, human, mouse, O. sativa(japonica cultivar-group), pan troglodytes, rat. This is the genome database of choice if it covers your organism as it contains minimal redundancy and the search speed is faster. Genome database (chromosomes from all organisms): Sequences from NCBI chromosome database (see BLAST database descriptions ) except that sequences whose accessions start with AC_ (alternate assemblies) are excluded to reduce redundancy. See BLAST database descriptions for information on other databases.
The larger the mismatches (especially those toward 3' end) are between primers and the unintended targets, the more specific the primer pair is to your template (i.e., it will be difficult to anneal to and amplify unintended targets). However, specifying a larger mismatch value may make it more difficult to find such specific primers. Try to lower the mismatch value in such case.
This specifies the size variation of the off-target PCR products relative to that of your intended PCR product. Only those primer pairs producing an off-target PCR product within the specified range will be tagged as non-specific.
If enabled, this program will NOT exclude the primer pairs that can amplify the mRNA splice variants of the same gene as your PCR template, thus making primers gene specific rather than transcript specific. This option requires you to enter a refseq mRNA accession or gi or fasta sequence as PCR template input because other type of input may not allow the program to properly interpret the result.
Maximum number of hit sequences (with unique sequence identifier) blast will generate for primer-blast to screen for primer pair specificities. Note that the actual number of similarity regions (or number of blast alignments) may be much larger than this number (for example, there may be a large number of alignments on a single hit sequence such as a chromosome).
Expected number of chance matches in a random model. Choose a higher value if you want more stringent specificity checking (i.e., to exclude primers that have even a large number of mismatches to unintended targets).
The maximum number of candidate primer pairs to screen in order to find specific primer pairs (The candidate primers are generated by primer3 program). Increasing this number can increase the chance of finding a specific primer pair but the process will take longer.
The number of consecutive Gs and Cs at the 3' end of both the left and right primer.
The maximum allowable local alignment score when testing a single primer for (local) self-complementarity and the maximum allowable local alignment score when testing for complementarity between left and right primers. See primer3 for details.
The maximum allowable 3'-anchored global alignment score when testing a single primer for self-complementarity, and the maximum allowable 3'-anchored global alignment score when testing for complementarity between left and right primers. See primer3 for details.
If the default "Automatic" setting is selected, the program will automatically select the repeat database using the following rules. 1. If a repeat database is available from the same organism as specified in the "Organism" field by user (see above), then that repeat database will be used. For example, if "Human" is specified, then the human repeat database will be selected. 2. If a repeat database from the same organism is not available, the database from the closest parent of that organism in the taxonomy tree will be selected. For example, the rodent repeat database will be selected if "Mouse" is specified in "Organism" field. However, no repeat database will be selected if "Gallus gallus" is specified since a repeat database from its taxonomical parents is not available.
The millimolar concentration of salt (usually KCl) in the PCR. Primer3 uses this argument to calculate oligo melting temperatures.
The millimolar concentration of divalent salt cations (usually MgCl2+ in the PCR). Primer3 converts concentration of divalent cations to concentration of monovalent cations using formula suggested in the paper Ahsen et al., 2001. [Monovalent cations] = [Monovalent cations] + 120*(v([divalent cations] - [dNTP])). According to the formula concentration of desoxynucleotide triphosphate [dNTP] must be smaller than concentration of divalent cations. The concentration of dNTPs is included to the formula beacause of some magnesium is bound by the dNTP. Attained concentration of monovalent cations is used to calculate oligo/primer melting temperature. See Concentration of dNTPs to specify the concentration of dNTPs.
The millimolar concentration of deoxyribonucleotide triphosphate. This argument is considered only if Concentration of divalent cations is specified.
Option for specifying the salt correction formula for the melting temperature calculation. There are three different options available: 1. Schildkraut and Lifson 1965, DOI:10.1002/bip.360030207 (this is used until the version 1.0.1 of Primer3).The default value of Primer3 version 1.1.0 (for backward compatibility) 2. SantaLucia 1998, DOI:10.1073/pnas.95.4.1460 This is the recommended value. 3. Owczarzy et al. 2004, DOI:10.1021/bi034621r
The nanomolar concentration of annealing oligos in the PCR. Note that this is not the concentration of oligos in the reaction mix but of those annealing to template. Primer3 uses this argument to calculate oligo melting temperatures. The default (50nM) 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.
Primer-BLAST A tool for finding specific primers