Re: how to clone regions of DNA binds to a specific protein ?

Hi "pra",

Thanks for your question. I was rather surprised to see this on the MadSci network, where we normally get questions from non-scientists. It sounds to me as though you are researchers in a professional institution, where I would have thought that there would be someone to advise you, or the facility to search the literature.

Anyway, you pose an interesting question. Normally, this problem is the other way around, in that people have a piece of DNA/RNA that they suspect binds to a protein and they wish to identify the protein. This problem is somewhat easier than yours, because the nucleic acid can be labelled, then exposed to an extract of soluble proteins to see which one binds. In your case, you need to know what DNA sequence, presumably from several promoter regions in the chromosome, is involved. Screening the chromosome, as it were, is somewhat more difficult.

I'd suggest 2 approaches. First, do you have any idea what sort of genes are activated by this transcription factor? If you do and any of them are cloned along with the upstream DNA, you can use this DNA in a gel mobility shift assay, ie see if the labelled DNA moves more slowly on a gel when exposed to the protein. To identify the precise binding region, you would have to go on to perform a DNA footprinting experiment, in which the DNA is labelled, incubated with the protein and digested, to see which region is protected from digestion. You ask how to clone the DNA binding region- remember that most binding regions are only bases to tens of bases in length, so cloning just the binding site is unlikely. You would need a larger piece of DNA containing the binding site for cloning.

Another approach which springs to mind is that of Lei et al (Journal of Cell Science 113:4523-, 2000), who synthesised a large pool of oligonucleotides and selected for those which bound best to a DNA-binding protein. But this is a rather expensive approach :-)

Finally, I suggest you get familiar with efficient literature search methods. Why not start at NCBI PubMed and try typing as keywords "DNA,binding,protein,identification" and whatever else you can think of. There are bound to be lots of good ideas there, if you sift through them.

Hope this helps, good luck in the research,

Neil