We’ve added a new feature (Max 3′ match), shown in Figure 1, to Primer-BLAST that limits the length of 3′ exon matches when designing exon-exon spanning primers. This makes it less likely that primers specifically designed to amplify transcripts will also amplify genomic DNA contamination in expression assays.
Figure 1. The new “Max 3′ match” option that limits the size of the 3′ match for exon-exon junction primers. This option helps avoid primers that may also produce product from genomic DNA.Primer-BLAST has always provided options to design transcript-specific primers that are useful for PCR-based expression assay. One option that you can use with a RefSeq mRNA accession as a template requires that primers span an exon-exon junction. These primers are more likely to amplify transcripts than the genomic sequence since the 5′ end and 3′ end of the primer binding site are split across an intron in the genomic sequence. However, if the 3′ match for a primer on an exon is too long, some primers may potentially amplify the exon from genomic DNA. The new option (Max 3′ match) added to the Exon/Intron Selection section of the Primer-BLAST submission form limits the length of the 3′ exon match for primers. The provided setting of 8 base pairs seems to give good results. You can adjust as needed. Setting this to a large value restores the previous behavior.
Added ability to limit max base match at 3 side of exon for primer-blast, to limit primer matches that can extend into an intron and possibly amplify .