Primer designing tool

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). Please enter the primer sequence only (No any other characters are allowed).

Optionally enter your pre-designed reverse primer. Always use the actual primer sequence (i.e., 5'->3' on minus strand of the template). Please enter the primer sequence only (No any other characters are allowed).

The Tm calculation is controlled by Table of thermodynamic parameters and Salt correction formula (under advanced parameters). The default Table of thermodynamic parameters is "SantaLucia 1998" and the default Salt correction formula is "SantaLucia 1998" as recommended by primer3 program.

With this option on, the program will try to return primers at the 3' side of template first. Note that obtaining such primers is not always possible due to other constraints (for example, there might be no unique region at the 3' side to generate target-specific primers).

This controls whether the primer should span an exon junction on your mRNA template. The option "Primer must span an exon-exon junction" will direct the program to return at least one primer (within a given primer pair) that spans an exon-exon junction. This is useful for limiting the amplification only to mRNA. You can also exclude such primers if you want to amplify mRNA as well as the corresponding genomic DNA.

Minimal and maximal number of bases that must anneal to exons at the 5' or 3' side of the junction Help

This specifies the minimal number of bases that the primer must anneal to the template at 5' side (i.e., toward start of the primer) or 3' side (i.e., toward end of the primer) of the exon-exon junction. Annealing to both exons is necessary as this ensures annealing to the exon-exon junction region but not either exon alone. Note that this option is effective only if you select "Primer must span an exon-exon junction" for "Exon junction span" option.

With this option on, the program will try to find primer pairs that are separated by at least one intron on the corresponding genomic DNA using mRNA-genomic DNA alignment from NCBI. This makes it easy to distinguish between amplification from mRNA and genomic DNA as the product from the latter is longer due to presence of an intron.

This specifies the range of total intron length on the corresponding genomic DNA that would separate the forward and revervse primers.

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.

Primer-blast tries to find target-specific primers by placing candidate primers on unique template regions that are not similar to other targets. However, in some cases, primer-blast cannot determine if a database sequence is an intended target or not, thus the user guidance might be helpful (For example, when your template is a polymorphic form or a partial region of an entry in the search database, or when the database such as the nr contains redundant entries of your template).
The "Automatic" option will ask for user guidance only when the program does not find sufficient unique template regions while the "User guided" option will always ask for user guidance if your template shows high similarity to any other database sequences.

Refseq mRNA:
    This contains mRNA only from NCBI's Reference Sequence collection

Refseq representative genomes:
    This database contains NCBI RefSeq Reference and Representative genomes across broad taxonomy groups including eukaryotes, bacteria, archaea, viruses and viroids. These genomes are among the best quality genomes available at NCBI. This database contains minimum redundancy in genome representation. For the eukaryotes, only one genome is included per species (However, alternate loci of eukaryotic genomes are included where applicable). For other species, genomes from diverse isolates of the same species may be included. Mitochondrion genomes are included where applicable.

core_nt:
    It is the same as nt except that it does not have the eukaryotic chromosomal sequences that are part of NCBI genome assemblies. The search speed is much faster than nt. We highly recommend using it over nt.

Refseq RNA:
    This contains all RNA entries from NCBI's Reference Sequence collection

Genomes for selected eukaryotic organisms (primary assemblies only):
   These are Refseq representative genomes from primary chromosome assemblies (i.e., no alternate loci) for many eukaryotic organisms. Mitochondrion and plastid genomes are also included where applicable.

Although sequences in this database are completely covered by the Refseq representative genomes database, it does not contain the alternate loci and thus avoids sequence redundancy introduced by including alternate loci. This database is recommended if you are not considering variations represented by alternate loci.

Custom:
   You can use your own sequences including nucleotide accessions, genome assembly accessions (such as GCF_000001635.27) or FASTA sequences as a search database. A maximum of 20 assembly accessions are allowed. FASTA sequences are limited to 300M. Note that the organism field is ignored for custom database.

This requires at least one primer (for a given primer pair) to have the specified number of mismatches to unintended targets. 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 more difficult to anneal to 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 is another parameter that can be used to adjust primer specificity stringecy. If the total number of mismatches between target and at least one primer (for a given primer pair) is equal to or more than the specified number (regardless of the mismatch locations), then any such targets will be ignored for primer specificity check. For examaple, if you are only interested in targets that perfectly match the primers, you can set the value to 1. You can also lower the E value (see advanced parameters) in such case to speed up the search as the high default E value is not necessary for detecting targets with few mismatches to primers.
Additionally this program has limit detecting targets that are too different from the primers...it will detect targets that have up to 35% mismatches to the primer sequences (i.e., a total of 7 mismatches for a 20-mer).
You may need to choose more sensitive blast parameters (under advance parameters) if you want to detect targets with a higher number of mismatches than default.

This specifies the max amplicon size for a PCR target to be detected by Primer-BLAST. In general, the non-specific targets become less of a concern if their sizes are very large since PCR is much less efficient for larger amplicons.

If enabled, this program will NOT exclude the primer pairs that can amplify one or more mRNA splice variants from the same gene as your PCR template, thus making primers gene-specific rather than transcript-specific (Note that it is NOT intended to generate primers that will amplify all variants. It only means that the primers may amplify one or more other slice variants, in addition to the one you have specified). Enabling this option will make it much easier to find gene-specific primers since there is no need to distinguish between splice variants. 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.