Posting form data to NCBI Primer-BLAST

Question

[Tom Harrop]

Hi all,

I'm trying to automate primer design for qPCR using NCBI's primer-BLAST web interface. My code is working fairly well but I've hit a problem with intron-less genes.

The form is at http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi. On the web form, clicking the "Get Primers" button posts your form data to http://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi:

<form name="searchForm" action="./primertool.cgi" enctype="multipart/form-data" method="post" class="f-wrap-1 " id="searchForm">

There is an input SPAN_INTRON, to enable or disable designing primers whose product will span an intron:

<label class="m" for="SPAN_INTRON">Intron inclusion</label>
    <div class="input "> 
        <input type="checkbox"  name="SPAN_INTRON" id="SPAN_INTRON"  class="checkDef" defval="unchecked" />                    
        <label class="right inlinelabel" for="SPAN_INTRON">Primer pair must be separated by at least one intron on the corresponding genomic DNA</label>
        <a class="helplink" title="help" href="#" id="intron_spanHl">[?]</a>            
            <p toggle="intron_spanHl" class="helpbox hidden">With this option on, the program will try to find primer pairs that are separated by at least one intron on the corresponding genomic DNA using mRNA-genomic DNA alignment from NCBI.  This makes it easy to distinguish between amplification from mRNA and genomic DNA as the product from the latter is longer due to presence of an intron.

    </div>

However this input seems to be ignored when posting to primertool.cgi. Here's a simple example: http://www.ncbi.nlm.nih.gov/tools/primer-blast/primertool.cgi?INPUT_SEQUENCE=NM_001185224&SPAN_INTRON=off returns the message:

No primers were found...see explanation below:  Exon/exon junction cannot be found for submitted PCR template.  Try search again without exon/intron requirements.

I've tried several values for SPAN_INTRON: off, false, False, unchecked, no and 0, but haven't had any luck. Does anyone know how to do this correctly?

Thanks for reading,

Tom

[Pierre Lindenbaum]

did you ever get a valid result ? do you handle cookies, redirection, POST method ?

[Tom Harrop]

Yes, it works fine for genes with introns. The initial POST returns a page with a job_key, which I am using to retrieve the BLAST results after waiting 60 seconds.

Answer 1

[Tom Harrop]

Revived this script two years later and worked out the SPAN_INTRON parameter. The trick is to post the form without SPAN_INTRON to turn the option off. Doubt anyone cares, but it may be useful for future me :)

[GenoMax]

Kudos for taking the time to come back and post a solution for a 2 yr old thread that now has a valid solution.

Answer 2

[807865101]

Hello，I also hit a problem like you。You must lick“Primer pair must be separated by at least one intron on the corresponding genomic DNA ”in intron inclusion,try again without the choice.