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.
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.
Will e-PCR for the command line be discontinued as well?
Yes, that is retired as well.
Thanks for your answer 🙁
Is there going to be a command line version of PrimerBLAST? Myself and other people in the community have used e-PCR for the command line in projects and pipelines. Thank you.
Thanks for asking. Currently Primer-BLAST isn’t portable. It would require significant changes and effort to make a standalone version. We’ll look into it.
Hi, It would be great at least to have a batch mode. e-PCR was great to see the hits on a chosen genome for a large set of primers at a time (10 if I recall). Now we have to do it one at a time. It’s a pain.
Thank you
Clara
I agree with you
I agree as well.
Please i have a big problem to deal with e-PCR using a mac os x ! can i get an alternative ? I have a lot of primers pairs to test into large genome files
Our current supported primer search tool is Primer-BLAST https://www.ncbi.nlm.nih.gov/tools/primer-blast/ You can search BLAST databases with primer pairs to identify targets and amplimer sizes.