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Primer-BLAST is the NCBI tool people usually mean when they search for NCBI BLAST primers, blast primers or primer blast NCBI. NCBI describes it this way: “Primer-BLAST was developed at NCBI to help users make primers that are specific to intended PCR target.” This guide walks through the form, the specificity settings, exon-exon junction primers, and how to handle the results.
What is Primer-BLAST and what is it used for in research?
Primer-BLAST combines two jobs that used to be separate. According to NCBI’s tool information page, it uses Primer3 (version 2.5.0) to design PCR primers and then uses BLAST and a global alignment algorithm to screen those primers against a database the user selects. The screen is meant to avoid primer pairs that can cause non-specific amplification, and it considers all pairings: forward-reverse, forward-forward and reverse-reverse.
Typical research uses include designing primers for a gene of interest from a RefSeq accession, checking whether an existing primer pair amplifies anything besides its intended target, and designing primers for qPCR or RT-PCR that are tied to a specific transcript. If you already have primers from a paper or a vendor, you can paste them in and let the tool check them. The general chemistry of the reaction itself is covered in our qPCR and RT-qPCR guide.
Install or access (official source only)
There is nothing to install. Primer-BLAST runs in the browser on NCBI’s site at ncbi.nlm.nih.gov/tools/primer-blast, and an information page describing the tool is at ncbi.nlm.nih.gov/tools/primer-blast/primerinfo.html. Use these NCBI addresses rather than third-party mirrors, so that the databases and the Primer3 version are the ones NCBI maintains. The published description of the method is Ye J et al., “Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction”, BMC Bioinformatics, 2012 (PubMed 22708584). NCBI’s information page suggests citing it as “Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction.”
How to use Primer-BLAST: step-by-step
The numbered steps below follow the section and field names on the current Primer-BLAST form. Defaults change over time, so the steps name the fields without quoting default values.
- Enter the PCR Template. Paste an accession, a gi number or FASTA sequence, or use “Or, upload FASTA file”. Use the Range From and To boxes to restrict design to part of the sequence.
- Optionally enter your own primers. The form has “Use my own forward primer (5′->3′ on plus strand)” and “Use my own reverse primer (5′->3′ on minus strand)”. Leave them empty to have the tool design primers.
- Set Primer Parameters. Here you set “PCR product size” (Min and Max), “# of primers to return”, “Primer melting temperatures (Tm)” (Min, Opt, Max and Max Tm difference) and “Primer Location Preference”.
- Choose exon and intron options if the template is a transcript. The Exon/intron selection area contains “Exon junction span”, “Exon junction match” (Min 5′ match, Min 3′ match, Max 3′ match), “Intron inclusion” and “Intron length range”. These are explained in the next section.
- Set the Primer Pair Specificity Checking Parameters. Tick “Enable search for primer pairs specific to the intended PCR template”, then pick the “Search mode” (Automatic, User guided or No user guidance), the “Database”, an “Organism” and, if needed, an “Entrez query (optional)”. Adjust “Primer specificity stringency” and “Max target amplicon size” as required.
- Review the Advanced settings. These include the self-complementarity limits, “TH: Max Self Complementarity” (Any, 3′) for the thermodynamic model and “Max Self Complementarity” for the older model, plus “TH: Max Pair Complementarity” and “Max Primer Hairpin”.
- Submit the form and wait for the results page to load.
How do I check primer specificity with Primer-BLAST?
Specificity checking is the feature that separates Primer-BLAST from a plain Primer3 run. On the form, the option is labelled “Enable search for primer pairs specific to the intended PCR template”, and its tooltip says the program will determine whether a primer pair can generate a PCR product on any targets in the database. The settings that shape the result are:
- Database. The choices listed on the form include Refseq mRNA, Refseq reference genomes, core_nt, Refseq RNA (refseq_rna), Custom and nt.
- Organism. The tooltip says this limits the specificity check to the organism you specify and that NCBI strongly recommends always specifying the organism when you are amplifying DNA from a specific organism. The field also lets you exclude organisms.
- Primer specificity stringency. Under “Primer must have at least” you set the total number of mismatches to unintended targets and the number of mismatches within the last base pairs at the 3′ end. The tooltip explains that at least one primer of a pair must have the specified number of mismatches to unintended targets. “Ignore targets that have” lets you disregard targets with a given number of mismatches.
- Search mode. With “Automatic”, the tooltip says the program asks for user guidance only when it does not find sufficient unique template regions.
If you are checking primers you already have, paste them into the own-primer boxes, set the organism, and read the target list on the results page as described below.
How do I design primers that span an exon-exon junction?
Junction-spanning primers are used so that a primer pair is more likely to amplify cDNA than contaminating genomic DNA. In Primer-BLAST:
- Use a transcript as the PCR Template, for example a RefSeq mRNA accession. The “Allow splice variants” option on the form notes that it requires a RefSeq mRNA sequence as the PCR template input.
- Under Exon/intron selection, set “Exon junction span” to “Primer must span an exon-exon junction”. The other choices are “No preference” and “Primer may not span an exon-exon junction”.
- Adjust “Exon junction match”. NCBI’s April 24, 2020 post on the Max 3′ match parameter explains that “Max 3′ match” limits the length of 3′ exon matches when designing exon-exon spanning primers. The post says a default of 8 bp seems to give good results, and that a large value restores the previous behaviour. The aim is that primers designed to amplify transcripts are less likely to also amplify genomic DNA contamination, because in genomic sequence the 5′ and 3′ parts of the binding site are separated by an intron.
- Alternatively, or in addition, tick “Primer pair must be separated by at least one intron on the corresponding genomic DNA” under Intron inclusion. Its tooltip says the program will try to find primer pairs separated by at least one intron on the corresponding genomic DNA. “Intron length range” sets the allowed intron size.
Output and how to read it
The results page lists the candidate primer pairs the tool returned (the number is set by “# of primers to return”). For each pair, check the following before ordering anything:
- Product size and location. Confirm that the product falls inside the “PCR product size” range you set and sits in the part of the template you intended.
- Melting temperatures. Check that both primers fall in the Tm window you chose and that the difference between them is within “Max Tm difference”.
- Specificity targets. When specificity checking is enabled, the results show whether the pair can generate products on other targets in the selected database. Any unintended target listed is a reason to pick another pair or tighten the stringency.
- Self-complementarity. The form exposes separate limits for “Any” and “3′” self-complementarity. In general terms, self-complementarity describes how well a primer can bind to itself, which is the route to hairpins and primer-dimers. Primer-dimers show up as a separate problem in melt-curve data; see qPCR melt curve analysis and primer-dimers.
What is self-complementarity in Primer-BLAST?
Self-complementarity is a design constraint. On the form, the Advanced settings include “TH: Max Self Complementarity” with “Any” and “3′” entries (thermodynamic model) and “Max Self Complementarity” for the older model. “Any” refers to binding between a primer and itself at any position, while “3′” concerns the primer’s 3′ end, which matters most because that end is where the polymerase extends. Primer-BLAST also offers “TH: Max Pair Complementarity” for the forward-reverse combination and “Max Primer Hairpin” for intramolecular folding.
If you report qPCR work, primer details belong in the methods; our MIQE reporting checklist covers what to record.
Troubleshooting
- The tool asks for guidance or finds too few candidates. In Automatic search mode the tool asks for user guidance when it does not find sufficient unique template regions. Choose “User guided” to steer the region, or widen the template Range and the product size limits.
- Unexpected off-target hits. Check the Organism and Database fields first. Without an organism the specificity check is not limited to your species of interest, and NCBI’s own tooltip recommends always specifying the organism for organism-specific work.
- Splice variants are excluded. By default the tool excludes pairs that amplify one or more mRNA splice variants. The “Allow splice variants” option turns this off and requires a RefSeq mRNA sequence as the template.
- Too few junction-spanning options. Revisit “Exon junction match”, and the Max 3′ match value in particular, as described above.
- Stringency. The stringency tooltip includes the advice to try a lower mismatch value; reduce it in small steps and re-check the target list rather than ignoring off-target hits.
Free alternatives
Primer-BLAST itself builds on Primer3. If you only need primer design without the BLAST specificity screen, the Primer3 project lists these options on primer3.org:
| Tool | What it is | Where it is described |
|---|---|---|
| Primer-BLAST (NCBI) | Primer3-based design plus BLAST and global-alignment specificity screening against a database you select | NCBI Primer-BLAST information page |
| Primer3Web | Web interface to Primer3, “a widely used program for designing PCR primers” | primer3.org navigation (primer3.ut.ee) |
| Primer3Plus | Another web interface to Primer3 | primer3.org navigation (primer3plus.com) |
| Primer3 (download) | The program itself, described as an open source, community-development project hosted by GitHub | primer3.org releases page |
Primer3 alone does not run the NCBI database screen, so a pair designed elsewhere is best checked for specificity by pasting it into Primer-BLAST as your own primers.
Related lab methods that depend on well-designed primers: site-directed mutagenesis, gene cloning and Sanger sequencing.
Frequently asked questions
What is Primer-BLAST?
It is an NCBI web tool for making primers that are specific to the intended PCR target. It uses Primer3 to design primers and BLAST plus a global alignment algorithm to screen them against a database you select.
How to use Primer-BLAST?
Enter a PCR Template, set the product size and melting temperature parameters, choose the database and organism for the specificity check, and submit the form. The step-by-step section above lists each field by its on-page name.
How do I check primer specificity with Primer-BLAST?
Enable the search for primer pairs specific to the intended PCR template, choose a database and organism, set the primer specificity stringency, and read the target list on the results page. Paste your own forward and reverse primers into the own-primer boxes to check a pair you already have.
What is self-complementarity in Primer-BLAST?
It is the design limit on how well a primer can bind to itself. The form offers separate Any and 3′ settings, in a thermodynamic model (TH: Max Self Complementarity) and an older model (Max Self Complementarity).
How do I design primers that span an exon-exon junction?
Use a transcript as the template, set Exon junction span to ‘Primer must span an exon-exon junction’, and adjust the Exon junction match fields, including Max 3′ match.
Are blast primers, primer-blast and NCBI primer blast the same thing?
These are common names for the same NCBI tool, Primer-BLAST. It is separate from standard BLAST sequence searching, which compares a sequence against a database but does not design primers.
Sources (official docs)
- NCBI Primer-BLAST tool form (field names read 2026-10-28)
- NCBI Primer-BLAST information page (read 2026-10-28)
- NCBI Insights, April 24, 2020: Max 3′ match in Primer-BLAST
- Primer3 project (read 2026-10-28)
- Ye J et al., BMC Bioinformatics 2012, PubMed 22708584
