High-quality primer design is essential for the success of all polymerase chain reaction (PCR)-based experiments. We previously developed a thermodynamics-based gene-specific quantitative PCR (qPCR) primer database for 147 organisms, which has been used extensively in gene expression studies. Howeve …

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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.

Primer-BLAST now offers direct links from genome assembly pages, so you can easily select the specificity database. Primer-BLAST also now accepts multiple target templates making it easy to design primers that can amplify several similar sequences such as all splice variants of gene or the same target (16S, COI) from different strains or species.