ChIP with unknown DNA target - (Aug/13/2007 )
I would like to do ChIP to see where my protein binds but I don't know what the targets are. I'd like to avoid using PCR to test a whole bunch of possibilities. Can I do ChIP then clone and sequence the isolated DNA? I feel like if this is possible I would have been able to find information about it but I can't find anything, also it would negate any need for ChIP on Chip so I think there is some reason this won't work but I can't think of it. Are there reasons why this won't work?
Find a lab with a solexa sequencing setup. I'm sure they would be fairly popular but you'd profit trying to do a colabotation with them. Then you can (quickly and cheaply) sequence all of the fragments you pull down in your IP. Here's a few references:
Science. 2007 Jun 8;316(5830):1497-502
Nature. 2007 Aug 2;448(7153):553-60
Otherwise ChIP on microarray (ChIP-chip) is your best (although more expensive and more difficult) alternative. One reason why cloning can be such a pain is the background you get from ChIP. You basically have to eliminate background to do a cloning study otherwise you can't tell whether a clone represents a real target or just background. Also, chromatin binding proteins (even proteins as specific as TFs) can have many targets so you have to get a really large number of clones.
you may do your Chip an digest DNA with dpn I (4site-restriction site. So i cut relatively high.
Then ligation of adaptaters, eventually PCR to amplify with primers which anneal on adaptaters, and then ligation.
My boss do that way.