DNA-protein interaction questions - a bit unusual - (Mar/08/2007 )

Hi

I have several questions regarding the in-vitro DNA-Protein interaction study i am trying to do:

Basically what i want to do is to incubate a transcription factor with the suspected promoter region, perform formaldehyde crosslinking, digest the unprotected DNA completely (to the level of single nucleotides or several nucleotide long molecules), extract the protein with the bound DNA, reverse the crosslink and extract the DNA for further analysis. I know that footprinting is a standard way to go, but i am trying to develop a slighlty different protocol (and avoid the sequencing reaction). I do not need to do any immunoprecipitation since the reaction will be performed in vitro and there will be no other proteins.

These are the questions i have:

1. How do I remove the formaldehyde from the reaction after the crosslinking is preformed ? Is dialysis the only way or there is some kind of wash protocol. If dialysis is the only way to go, what kind ? Using bags or rings?

2. What enzyme should i use to digest the exposed DNA ? As far as i understand DNAseI does not digest as long as there is DNA to work on. Or maybe it only depends on the incubation time ?

3. What is the best way to reverse the crosslink ? Proteinase K? I prefer not using protease, since this would force me to perform an additional protease neutralization reaction.

4. In case i am not using protease, how do i separate the DNA from the protein ? Will regular ethanol precipitation protocol work with this relatively short sequence (i expect the transcription factor to protect anywhere between 12-22 bases).

Thanks

Dunno much about 1, 3 and 4, but I think for 2.) Nuclease P1 should be good.

I used a combination of Alkaline Phosphatase and Nuclease P1 to digest DNA into nucleosides.
Leaving away the Alkaline Phosphatase and using only Nuclease P1 should yield the desired nucleotides, instead of the nucleosides I got.
For a complete digest be sure to use excess amount of enzyme. Can't remember how much I used (althoug I think sth. like 1U for 20 µg of DNA in total vol of 50µl, but I will have to check).

OK, so here are some of the answers i found, hoping they would work.


Formaldehyde needs to be quenched first with 4M glycine solution. After that I will try to digest the DNA with DNAseI for 30 mins at 37 degrees. Then i will neutralise the DNAse by incubation at 75 degrees for 5 mins and then reverse the crosslink by incubating at 65 degrees for 6 hrs. Then i am gonna do ethanol/chloroform DNA precipitation

I will write if it works, maybe someone sometimes will find it useful.