Primer designing tool

PCR Template

Enter accession, gi, or FASTA sequence (A refseq record is preferred) [?] Clear

Enter the PCR template here (multiple templates are not supported at the present time). A RefSeq accession or GI will allow Primer-BLAST to use information like gene identity, transcript variants and SNP features to perform better primer specificity checking as well as to avoid SNP sites.
A template is not required if the forward and reverse primers are entered below.

Or, upload FASTA file

Range

From To

Forward primer

Reverse primer [?]

Enter the position ranges if you want the primers to be located on the specific sites of template sequence. Note that the position range of forward primer may not overlap with that of reverse primer.

Primer Parameters Use my own forward primer

[?]

Optionally enter your pre-designed forward primer. Always use the actual primer sequence (i.e., 5'->3' on plus strand).

Use my own reverse primer

[?]

Optionally enter your pre-designed reverse primer. Always use the actual primer sequence (i.e., 5'->3' on reverse strand).

PCR product size

Min Max

# of primers to return

Primer melting temperatures (T_m)

Min Opt Max Max T_m difference

Primer Pair Specificity Checking Parameters Specificity check

Enable search for primer pairs specific to the intended PCR 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 orientation. 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.

Organism

Database

[?]

The genome database contains 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. For other databases, please see BLAST database descriptions .

Primer specificity stringency

At least total mismatches to unintended targets, including
at least mismatches within bp region at the 3' end [?]

The larger the mismatch value to the unintended targets is, the less likely a primer pair will anneal to off-target templates and generate a PCR product (therefore the primers are more specific to your target). However, a larger mismatch value may make it more difficult to find such specific primers. Try to lower the mismatch value in such case.
Locations of mismatch between the primer and the template also matters for PCR. Mismatches close to the 3' end of the primer will have much more adverse impact on primer extension than those toward the 5' end.

Misprimed product size deviation

[?]

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.

Splice variant handling

Allow primer to amplify mRNA splice variants (requires refseq mRNA sequence as PCR template input) [?]

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.

Show results in a new window

Advanced parameters

Primer Parameters Primer Size

Min Opt Max

Primer GC content (%)

Min Max

GC clamp

[?]

The number of consecutive Gs and Cs at the 3' end of both the left and right primer.

Max self complementarity:

[?]

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.

Max 3' end complementarity:

[?]

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.

SNP handling

Primer binding site may not contain known SNP

Repeat filter

Avoid repeat region for primer selection by filtering with repeat database

Concentration of monovalent cations

[?]

The millimolar concentration of salt (usually KCl) in the PCR. Primer3 uses this argument to calculate oligo melting temperatures.

Concentration of divalent cations

[?]

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.

Concentration of dNTPs

[?]

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

Salt correction formula:

[?]

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

Annealing Oligo Concentration

[?]

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.

Internal hybridization oligo parameters Hybridization oligo

Pick internal hybridization oligo

Hyb Oligo Size

Min Opt Max

Hyb Oligo tm

Min Opt Max

Hyb Oligo GC%

Min Opt Max

Show results in a new window