qPCR primer design

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science
science's picture
qPCR primer design

I am interested in designing primers to determine amplification or deletion of my gene in the genome. On e-PCR (NCBI) I get results in my "genomic section" of the same species I am interested in. Now most of these seem to locate to the same chromosome, however, I was wondering how I determine whether they are infact of my gene of interest, or other genes targeted by my PCR primer set. It does not show any results in the "cross-references" section that is not the orginal gene of interest.

However, I do have another gene that does show "cross-reference" to another gene in all primers designed by e-PCR. What would be an appropriate methodology for designing primers for this gene. Thank you.

Real Time PCR
Real Time PCR's picture
I would suggest that you go

I would suggest that you go for primer design that is specific to your gene and avoids other genes in your chromosome. Use AlleleID or Beacon Designer software. They automatically interpret the homology with a database (that you have to specify). You can also create a custom database by downloading all the sequences in your chromosome locally and then BLASTing using Beacon Designer. The algorithm of the software will avoid regions that are similar between your gene and other genes.

Set it up first and then ask for a trial to the Bioinformatics company
that authors the software. Here is a direct link to Beacon Designer website
http://www.premierbiosoft.com/molecular_beacons/index.html

the software price is a bit steep at $2585. You can ask them if they will design these sort of primers for you. They do IMO.

Jen_Floyd
Jen_Floyd's picture
I use the Harvard Primer Bank

I use the Harvard Primer Bank for all of my qPCR primers.

http://pga.mgh.harvard.edu/primerbank/

I normally order 2-3 sets of primers and then test them for similar amplification efficiency with my control primers and also check for single product with a dissociation curve.

If you're not doing mouse or human qPCR try Perl Primer

http://perlprimer.sourceforge.net/

You can blast the resulting primers to make sure they are unique. Also always do dissociation curve on new primers (or run your products on a gel) to make sure they're truly unique.

All this is free btw.

Good luck!

R Bishop
R Bishop's picture
This topic has been moved to

This topic has been moved to the qPCR Forum

Jen_Floyd
Jen_Floyd's picture
 When I design primers for

 When I design primers for QPCR, I generally use the Harvard Primer Bank (see above).  This may not work for you since it sounds like you're trying to amplify from genomic DNA but you could check them to see if they cross a intron-exon boundary or not.  Otherwise, I would use the Perl Primer (see above) and choose QPCR primers for the primer type and make sure they stay within one exon.  This should give you many primer choices and you can BLAT (you cannot BLAST primers because they are too short) each primer set to make sure it is not found somewhere else in the genome.  Once you have at least three primer sets for each gene (don't forget to include a control) you are ready to test the primers.
Do a standard curve for each primer set with some control DNA in a 1/3 serial dillution (I usually do three primer sets for each gene).  Include a dissociation curve in the thermocycling profile to insure you have only one product (strange things sometimes happen to PCR reactions).  Once you have your Ct values, plot the log of the dilution vs the dCt (subract the Ct for your control gene from the Ct for your gene of interest).  Make a dCt for each primer set pair so for each primer set you should get at least three dCts and therefore three plots.  Choose the primer set pair that has a slope absolute value less than 0.1 (close to zero).  This will allow you to do the rest of your QPCR using the ddCt method of quantitation and will save you time and money over the standard curve method.
If your gene is particularly repetitive, it's possible that you might need to troubleshoot this a bit.  If you can amplify within an exon, I think it will avoid that issue.  Primers are relatively cheap and easy to order (we get ours from IDT for a couple of bucks with next day service).  I do RT-QPCR on a regular basis with Sybr Greener from Invitrogen and this method of choosing primers has been very helpful for me.  I think with some minor modifications it will work for genomic DNA quantification.
Best of luck!

Ivan Delgado
Ivan Delgado's picture
This is the first time I've

This is the first time I've heard of the Harvard primer bank. Sounds like a great resource. 
According to the website the ~27,000 primer pairs tested so resulted in a success rate of almost 83%, which is pretty good considering the amount of time this can save you. 
My question is: anybody out there have any experience with these primers? The "success rate" seems to be based on visualizing a band on an agarose gel. This does not seem to be very convincing to me as far as their usefulness in real time PCR experiments. 
Any feedback is appreciated.

Jen_Floyd
Jen_Floyd's picture
I have been using Harvard

I have been using Harvard Primer Bank almost exclusively to pick QPCR primers.  They give you a lot of options and since time is more important that the cost of primers I usually order three sets and do my standard curve vs three sets for b-actin to look at amplification efficiency.  I don't run them on a gel but do the dissociation curve and I would say that maybe 1 in 10 has more than one PCR product by that measure.
 
Ivan wrote:

This is the first time I've heard of the Harvard primer bank. Sounds like a great resource. 
According to the website the ~27,000 primer pairs tested so resulted in a success rate of almost 83%, which is pretty good considering the amount of time this can save you. 
My question is: anybody out there have any experience with these primers? The "success rate" seems to be based on visualizing a band on an agarose gel. This does not seem to be very convincing to me as far as their usefulness in real time PCR experiments. 
Any feedback is appreciated.
Ivan Delgado
Ivan Delgado's picture
Thanks for the feedback. I

Thanks for the feedback. I could not agree more that testing three primers at a time to make sure you get an assays that works is a great time saver. I will definitely take a look at this resource next time I need to design primers. 

calou80
calou80's picture
I am suggesting this web

I am suggesting this web interface: http://updepla1srv1.epfl.ch/getprime/

You could directly enter your gene of interest and get the best primers (specific to a transcript or covering all the transcript of your gene). It is very easy and give good primer quality.

Cheers