I have gene having two variants (SNP). The case is compound heterozygous i.e one variant is maternal and other is paternal. One variant is on exon and another is on promoter site of the gene. This variant on promoter site is 7223 base pair upstream of codding sequence. Can anyone please suggest an assay to check impact of this point mutation on promoter site. Or any software by which I can check which transcription factor is binding to this promoter. For the exon variant I have already checked its impact on protein.
Prediction of transcription factor binding in silico is not generally reliable. The reason is that there are more factors influencing this binding than just sequence (e.g. cooperative binding of factors, higher order structure of chromatin). So, unfortunately the only way to find out whether your SNP influences transcription factor binding or promoter activity is to measure these directly.
The normal method for measuring promoter activity is to clone the promoter into an vector to drive luciferase or another reporter gene (eg. pGL3). Then transfect the mutant and wild-type luciferase constructs into a cell line (generally one where your gene of interest os normally expressed) and measure luciferase activity.
To measure the effect of the SNP on transcription factor binding you will need to do mobility shift assays (which are hard work).
If you have access to RNA where your gene of interest is expressed then an easier method may be to look at the expression of your gene in a patient or relative heterozygous for the promoter SNP. If you can find another SNPin the RNA then you can look for allelic expression imbalance using the second SNP as a marker for each allele. If your promoter SNP affects expression you should see a bias away from a 50:50 level of expression in the second SNP.
Best of luck.
Steve
Consider trying EMSA technique to analyse your promoter. It may help in this.
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