Electronic-PCR (e-PCR) is retiring, use Primer-BLAST instead


NCBI is retiring the e-PCR tool effective immediately. The good news is that an existing tool, Primer-BLAST, fills in nicely for the functions of both Forward and Reverse e-PCR, and has the additional benefit of de novo primer design.

primerblast

What can Primer-BLAST do?

  • Design primers for your template sequence.

This is similar to using Forward e-PCR to locate STS markers in the provided sequence. It uses the latest version of the primer3 program, and allows adjustment of most of the same primer3 parameters.

  • Check the specificity of designed, or supplied, primers.

This is similar to using Reverse e-PCR to search a sequence database with STS markers. For current users of primer3, you do not need to run a separate BLAST search after you’ve designed your primers. Primer-BLAST automatically searches a user-selected database for your organism, or group of organisms, and it provides a nice graphical view of the primer positions.

How flexible is Primer-BLAST?

It is often unnecessary to change the default settings, but here is a selection of the many parameters you can adjust:

  • When your template is an NCBI RefSeq mRNA (NM_ or XM_ accession), Primer-BLAST can:
    • include or exclude exon-exon junctions,
    • require that primer pairs are separated by at least one intron, and
    • find primers that amplify splice variants, not just the input template.
  • You may also adjust:
    • PCR product size
    • primer melting temperatures
    • whether or not to exclude primer binding sites that contain known SNPs
    • BLAST settings
    • and many more parameters.

Give Primer-BLAST a try

You can link to Primer-BLAST from the “Specialized searches” section of the BLAST home page.

For help getting started, see the Primer-BLAST factsheet, and a short, 4 minute demonstration of Primer-BLAST beginning at 50m:13s in this video, “Webinar: A Practical Guide to NCBI BLAST”. Finally, don’t overlook the more than 40 in-page tips that help explain the parameters; use the question mark icons to reveal those tips.

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