funtional significance of promoter SNP - (Aug/12/2005 )
we found a SNP in the promoter region of a gene and strong association between thhis SNP and disease was found. so, how to evaluate the funtional significance of one SNP in the promoter of a gene? I need detailed protocol or stratege. thank you!!!!
well don't get completely what you mean, but you can first check mRNA and protein levels, and if targets are known, do an analysis of their quantities and eventually of their phosphorylation state...
I think this is a rather general question that you are asking, by general I mean that answers to your question could not be as simple as a step-by-step protocol. The reason is that a lot of detailed information is needed to decide which way to go, for example, what is the gene, what is the disease, what is the protein product the gene is coding, is it a kinase, a phosphatase, or a transcription factor?
These being said, I think we can only give you a general advice as a guide for how to do it if we were in your research position.
I think there are at least 2 directions to go at the same time:
1. It is a straight forward question to ask that how is the SNP in the promoter affect the expression level of the gene, is mRNA level and protein level affected and how much?
2. Is the gene product directly or indirectly related to the disease. Here information about the disease pathway, the gene product and papers that links these two are very important.
After having some idea about the 2 questions, then I think you can start out to crack the key problem identified in this process.
Since the SNP is located in the promoter region you would like to see if it has any expressional function. So what you need to do is a transcient transfection assay in a relevant cell line under relevant circumstances. If the SNP increases or decreases the expression then you should hit the internet figuring out if any transcription factors are likeliy to bind into your area and if the SNP affects this binding. If a potential binding site is created or lost then design a Chromatin IP experiment or a blot where you can look specificially on if the SNP actually has a binding disturbance.
Anyway it is difficult to point out a specific strategy based on the information that you provided, but I hope this helped some