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).