design primer for chip - (Sep/10/2009 )

Hi all,

i'm trying to design a qPCR primer for the sequence where PPAR alpha binds onto the mice fads2 promoter to amplify the ChIP DNA product.

i'm a work-study student and i have no idea how to do this... can some one help me find what the sequence that ppar-a bind to fads2? or at least what site to use/start?

please help!

thank you,
confused

xyz123 on Sep 10 2009, 04:57 PM said:

I've found the Evolutionary Conserved Region Browser (ECR Browser) to be of use when trying to orient myself in the genome:

http://ecrbrowser.dcode.org/

After searching for your gene of interest it can also return transcription factor binding sites and their evolutionary conservation (click synteny/alignments after you have your sequence lined up in the browser). Either way, this is a good place to start and get a handle on what your gene looks like etc...

After you figure out where your promoter region is then you can use NCBI "gene" to get the appropriate sequence, take it to your favorite primer design site (e.g, Primer BLAST) and have at it.

thanks for the reply mighty mouse, but it didnt give me any transcription factor binding sites (or options to select them) after i clicked the synteny/alignment link. all it did was give me homologous sites on other species.

i guess to simplify what i need.

1. find promoter region of mouse fads2 gene
2. find transcription binding sites on the promoter
3. find specific site (sequence) that ppar alpha bind
4. design primer for that particular sequence.



1. found on USCS
2. dont know where to start looking for this... the ecr site might mouse suggested didnt give transcription factor binding sites. ?:/

3. i think i found a couple of binding site sequences on the genomatix site, but it talks about matrix family assignment. i used the 2 binding site sequences for PPAR_RXR matrix (is this the same thing as transcription binding sequence?) from the site to scan 2000bp upstream of gene with no luck.

4. TBD

please help.
still confused

Hi xy,

So to get the transcription factor binding sites (TFBS), line up the presumed promoter region in the ECR browser, then click synteny/alignments as you did. A screen will come up asking you which other species you are interested in looking for conserved binding sites in. Click on the "Mulan" link on the right for the species your interested in comparing your species to under the "Alignment/TFBS" heading. A new window should open up. Then click through several windows (submit....continue...MutliTF...submit....select all (or just the TF your interested in) then submit...) then at the end you'll get to a screen where you can look at conserved TFBS between the two species previously selected, or at the bottom they have a link to all TFBS with the option to look at either of the two species you previously selected.

I hope that helps!

Why don't you just design 3-4 different primer pairs covering the region around the predicted TSS, and try them all. If you see an enrchment of one of the fragments, then you have found your binding site. Very simple.

check http://bioinforx.com/web/product_bxchipseq.php for more details.

Let me know if it helps. Thanks