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Maximum number of database sequences (with unique sequence identifier) Blast finds for primer-blast to screen for primer pair specificities. Note that the actual number of similarity regions (or the number of hits) may be much larger than this (for example, there may be a large number of hits on a single target sequence such as a chromosome). Choose a higher value if you need to perform more stringent search.
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Expected number of chance matches in a random model. A higher E value should be used if you want more stringent specificity checking (i.e., to identify targets that have more mismatches to the primers, in addition to the perfectly matched targets). On the other hand, a lower E value is recommended if you are only interested in perfect or nearly perfect matches as this will significatly shorten the search time.
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The minimal number of contiguous nucleotide base matches between the query sequence and the target sequence that is needed for BLAST to detect the targets. Set a lower value if you need to find target sequences with more mismatches to your primers. However this will increase the search time.
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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.
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The maximum number of PCR targets (amplicons) to be shown when designing new primers.
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The maximum number of PCR targets (amplicons) to be shown when checking specificity for pre-designed primers.
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The maximum number of PCR targets (amplicons) to be found on any single sequence in the search database.
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The number of consecutive Gs and Cs at the 3' end of both the left and right primer.
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The maximum allowable length of a mononucleotide repeat, for example AAAAAA.
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The maximum stability for the last five 3' bases of a left or right primer. Bigger numbers mean more stable 3' ends.
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The maximum number of Gs or Cs allowed in the last five 3' bases of a left or right primer.
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The option "Use Thermodynamic Oligo Alignment" instructs Primer3 to use thermodynamic alignment models (instead of old traditional secondary structure alignment) for calculating the propensity of oligos to form hairpins and dimers while the option "Use Thermodynamic Template Alignment" instructs Primer3 to use thermodynamic alignment models (instead of old traditional secondary structure alignment) for calculating the propensity of oligos to anneal to undesired sites in the template sequence.
(For thermodynamic alignment model only)
(For thermodynamic alignment model only)
(For thermodynamic alignment model only)
(For thermodynamic alignment model only)
(For old secondary structure alignment model only)
(For old secondary structure alignment model only)
(For old secondary structure alignment model only)
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E.g. 401,7 68,3 forbids selection of primers in the 7 bases starting at 401 and the 3 bases at 68. Or mark the source sequence with < and >: e.g. ...ATCT<CCCC>TCAT... forbids primers in the central CCCC.
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Enter a list of space separated nucleotide positions. This requires that the left or the right primers to span a junction that is just 3' of any such positions. For example, entering "50 100" would mean that the left or the right primers must span the junction between nucleotide position 50 and 51 or the junction between position 100 and 101 (counting from 5' to 3'). You can also specify in the fields below the minimal number of nucleotides that the left or the right primer must have on either side of the junctions. This option is useful if you want a primer to a span specific junction on the template. Note that this option cannot be used in association with the "Exon/intron selection" options above.
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The millimolar concentration of salt (usually KCl) in the PCR. Primer3 uses this argument to calculate oligo melting temperatures.
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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.
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The millimolar concentration of deoxyribonucleotide triphosphate. This argument is considered only if Concentration of divalent cations is specified.
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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 ofPrimer3 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
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Option for the table of Nearest-Neighbor thermodynamic parameters and for the method of melting temperature calculation. Two different tables of thermodynamic parameters are available:
Breslauer et al. 1986, DOI:10.1073/pnas.83.11.3746 Inthat case the formula for melting temperature calculation suggested by Rychlik et al. 1990 is used.
SantaLucia 1998, DOI:10.1073/pnas.95.4.1460 This is the recommended value.
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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.
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With this option on, the program will automatically retrieve the SNP information contained in template (using GenBank accession or GI as template is required) and avoid choosing primers within the SNP regions.
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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.
Avoid repeat region for primer selection by filtering with repeat database
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Low complexity regions are some regions in a DNA sequence that have biased base compositions such as a stretch of ACACACACACACACACACA.
