How to find the promoter region of a gene for methylation study - (Feb/03/2005 )
This is the URL to my gene of interest.
I am trying to estimate the promoter region of this gene so that I can do Meth specific PCR on the promoter..the promoter region of this gene has not been positively identified..but the exon information is known. I am new to this field.. Could anyone please help me out, preferably using step by step directions if possible.
Here is what I did to find the 5' flanking sequence of your gene:
1. I searched NCBI nucleotide database using "gene name + promoter" or "gene name + flanking" and got no hit.
2. Then, I went to ensembl.org and found the gene, clicked "View genomic sequence for this gene with exons highlighted", and asked it to export 1kb 5' sequence.
3. I copied the 5' sequence and fed to methprimer to predict CpG islands. Luckily, there is a CpG island close to the first exon (see fig below), which means the 5' sequence is likely the promoter of the gene.
4. I pasted the sequence back to NCBI blastn program to see if it matches experimentally identified promoter sequences that I might have missed by gene name text search. In this case, I didn't find any, which means the promoter of this gene has not been characterized yet. You can safely assume the sequence I got is the 5' regulatroy sequence and go ahead with methylation mapping of the identified CpG island.
Hope that's helps.
5' Flanking sequence (red is exon):
CpG island prediction (blue) and BSP primer design
Thank you very much!! I have one more query..Is there a way to do this using the Entrez gateway? The problem is my boss trusts ncbi more than anything else..thank you very much again!
You can just blast NCBI database using the sequence I got from ensembl. I am sure this sequence and the mRNA sequence will align to the same genomic sequence. Actually all genome databases (ensembl, UCSC and NCBI) use the same data and assembly but present the data differently. ensembl provides the best genome browser.