Design construct for promoter activity assay - (May/23/2013 )

Hi all,
I am going to check and compare the promoter activity of 2 different SNPs.
Thus, I have to design two constructs for cloning.
What should we consider when we design the construct for cloning so that we can get the promoter activity form them?

Thank you very much and hope you can help me...

-DrLeo-

If you have only one SNP to study, you can have two constructs, each contains one allele of the same promoter sequence. The insert should include some extra sequence upstream of the SNP, and downstream of the TSS.

since you have two SNPs in the promoter, the number of constructs needed depends on how many haplotypes you observed in population. The constructs should simulate the haplotypes.

You can amplify the desired inserts from DNA with the same alleles and then clone the products into a reporter vector.

-pcrman-

Thanks pcrman,
I still do not know how can I get the construct. Do I have to include any special sequence from the promoter region together with my SNPs?

-DrLeo-

You can genotype DNA from cell line or any individual to find homozygoze or heterozygote for the SNPs, then you design primers to amplify the promoter region including the SNP, and clone the product into a reporter vector. You can take a look at this paper. Beware that this paper dealt with a single SNP in promoter.

-pcrman-

Thanks pcrman,
As I know, in one gene, there is just only start position for transcription. Am I right?
How can we determine the start position and the promoter region exactly?
Thank you again!!!

-DrLeo-

in one gene, there is just only start position for transcription. Am I right?

That is not always right. Many genes have more than one TSS.

Please take a look at this post about how to find promoter sequence and determine TSS

-pcrman-

Thank you very much, pcrman

-DrLeo-