Chromatin Remodelling / restriction enzyme accessibility - (Mar/03/2009 )
This may seem like very simple questions but I am new to this. I have two cell lines in which I wish to compare the chromatin accessibility of the promoter region of my gene of interest (papers have indicated that this gene can require chromatin remodelling in the promoter region to become accessible).
So my supervisor has told me to check if this applies in the cell lines I am working with. I have been reading up as much as I can on the topic but am still unclear on many things so any help is appreciated.
As i understand from the methods of the paper i mentioned the methodology is
1. Isloate nuclei 2. Treat with desired enzyme 3. Klenow treatment 4. Ligation of DNA to a universal linker 5. Cleavage sites detected by PCR - with long universal primer & gene specific primer. 6. PCR products labelled with radioisotopes, further PCR cycles & visulaisation on a gel.
Firstly, the universal linker - where exactly does this fit in? and the universal primer is it to attach to the linker?
zzzzzzzzzzzzz ctc att g CUT gt ccc zzzzzzzzz OR if restricted zzzzzzzz ctc att ggt ccc zzzzzzzz
Secondly, can i avoid the labelling with radioisotopes as I am not trained (thus not allowed) to work with them at the minute.
Thanks in advance
You are doing in vivo footprinting using ligation medated PCR (LM-PCR). Probably you want to read these papers
http://genome.cshlp.org/content/2/2/107.full.pdf Analysis of chromatin structure by ligation-mediated PCR
http://web.nchu.edu.tw/pweb/users/sdai5497/research/2356.pdf Ligation-mediated PCR for quantitative in vivo footprinting
I believe you have to use isotope for such experiment.