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Primer-BLAST Results Help
Primer-BLAST was developed at NCBI to help users make primers that are specific to intended PCR target. It uses Primer3 to design PCR primers and then uses BLAST and global alignment algorithm to screen primers against user-selected database in order to avoid primer pairs (all combinations including forward-reverse primer pair, forward-forward as well as reverse-reverse pairs) that can cause non-specific amplifications. To cite Primer-BLAST or look for more details, please consult our publication:
Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden T (2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics. 13:134. Use of Primer3 itself is subject to the terms and conditions stated on Primer3 web site. Explanations of fields in primer report. Tm: This is the melting temperature for primers. GC%: This is the percentage of G and C bases in a primer. Self complementarity: This describes the tendency of a primer to bind to itself (therefore interfering with target sequence binding). It will score ANY binding (using local alignment) occurring within the entire primer sequence. The lower this value is, the less likely the primer will bind itself, which is preferred. Note that there are two options on Primer-BLAST interface to calculate this value (traditional secondary structure model or thermodynamic model). Self 3' complementarity: This is similar to the above "Self complementarity" except this describes the tendency of a primer to bind to itself toward the 3' end. Additionally, it is calculated using global alignment rather than local alignment. The lower this value is, the less likely the primer will bind itself toward 3' end, which is preferred. There are also two options to calculate this value (traditional secondary structure model or thermodynamic model).
Exception error: No sequence input was provided.
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If you want to allow any of the unintended targets, check the box(es) next to the ones you accept and try again to re-search for specific primers Help
Primer-BLAST can only find non-specific primers for your template since there are other targets that are similar or identical to your template. You may choose to re-search for specific primers by allowing some of the highly similar off-targets if they are irrelevant to your PCR experiment. For example, not all transcript variants are expressed in all tissues, or you are not concerned about the predicted transcripts (i.e., XM_ accessions), or there are redundant database entries. Another example is, when you enter a raw sequence as PCR template rather than an accession, Primer-BLAST will likely report many off-targets. However, some of these might be your intended target that you should choose to allow. When Primer-BLAST does not need to avoid the highly similar off-targets in selecting unique primer regions, it typically has a much higher chance to find specific primers for your intended target.
