I want to search for any and all amplicons that could be produced from my primer pairs. My template will be DNA from human stool samples. My primers are intended to amplify specific microbes, but I want to see if there are any other unintended targets in NCBI database. I can't seem to find the correct settings, I'm using Primer-BLAST and always the results are only from my intended target. Ideally, I'll be able to exclude my intended organism from the search. Amplicons of ANY size are very relevant.
Anyone have knowledge or experience with this? Thanks!!!
Hi Melissa,
I'm afraid all of what you're asking is unaccomplishable using primer-blast. The whole NCBI refseq database is too big (no, really) to search through. You can put multiple specific organisms under "organism" field though by clicking "add organism".
You can even use a bigger taxon (for example Mammalia of Bacteria) if you switch to "refseq representetive genomes" database in the "database" dropdown list, and then specifying a taxon in "organism". But be prepared to wait quite some time.
If you want to exclude an organism you must put a query in an "entrez query" field. For example if I want to exclude e.coli, then "exclude: Escherichia coli (taxid:562)" must be written in the field. "exclude: species name (taxid:species id)" in general.
Taxids can be found here: https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi.
You can also start typing your unwanted organism beforehand in the "organism" field, autocomplete it and then just copy from there instead of searching and then replace it with your "bacteria" taxon or whatsoever.
Amplicon size can be specified under "Max target amplicon size", but your polymerase might not be able to amplify the fragments that are too long (typically for regular Taq it's a 2-3kb cutoff)
Hope this answers your question
Hi Melissa
at your place I would first product the amplicon and blast its sequence against all organism. Use the UCSC web server to test in silico your PCR (http://genome-euro.ucsc.edu/cgi-bin/hgPcr?hgsid=284774770_oBgAkvOWQO8wLFJ9RRbHTtNYEoFk), get the sequence of your amplicon and submit it in the NCBI blast to get what you're searching for.
all the best
fred
Dirk Schmidt
, you're exactly right. I only get hits for my pathogen of interest. I'm not able to see where I can exclude an organism in Primer-Blast. I've definitely used it to exclude in Blast-n. I suppose it's possible that my primers don't bind anything except my pathogen of interest.....but this would surprise me! Thank you!
I just finished a webinar with NCBI covering Primer-BLAST. It is possible to search for potential targets for 2 primers.....but it's not perfect! ie. my 2 primers match a few off target sequences in the database, and the length is provided, but my PCRs are showing many other amplicons. SO, it's a place to start i guess. Additionally, you can do a normal Blastn search using query sequence with your 2 primers at the ends and your probe seq in the middle with 20+ "N"s flanking the probe seq. It also "kind of" works. Basically, the original answer from Leonid Ilchuk was accurate; it's not accomplishable! So now I'm just amplifying and sequencing all results, then designing a target specific probe that will hopefully eliminate all other products.
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