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I'm interested in sequencing and analyzing the bound DNA, and minimizing the amount of unbound DNA that gets sequenced through digestion.

When digesting protein-bound DNA, is all of the unbound DNA digested? Is there a way to maximize the amount of unbound DNA that is digested?

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4 Answers 4

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Well, it depends if you just want to sequence your protein bound fragments. If that is the case, I suppose you could go for a FAIRE-seq. Basically, you perform several iterative phenol/chlo extractions, thus separating the bound and unbound fragments. Then you sequence the unbound fragments that are present in the aqueous phase, and after the mapping, you can deduce the regions that were actually bound to proteins.

I don't know if it is possible to treat your bound fragments (that you isolate after a phenol/chlo extraction in the organic phase) with proteinase K, then perform another phenol/chlo extraction to purify your formerly bound fragments and sequence them. Could be worth trying.

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This is a difficult experiment. It's usually done by digesting the DNA under conditions where the binding protein is solidly attached. DNAse is added, and then after some time, the DNAse is degraded with heat or the addition of an inhibitor.

What you get depends on the length of the DNA protected by the protein and the tenacity by which the protein binds itself to the DNA.

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Are you interested in transcription factor binding (and similar), or in histone modifications?

If you are looking at investigating something with a short binding motif that occurs redundantly across the genome, ChIP-exo works: http://www.cell.com/abstract/S0092-8674(11)01351-1

This technique involves digesting one strand of the DNA up until the bound protein-DNA complex via exonuclease, while leaving enough of a tag on the other end to uniquely identify it's location within the genome. I have no first-hand experience with it, and it's yet to be independently replicated, but it looks fantastic. I don't recall whether they investigated the extent to which off-target DNA was digested by the exonuclease, but theoretically I expect a significant portion would have been eaten up.

If you are interested in ChIP of histone modifications, you can use micrococcal nuclease (MNase) for your sample prep instead of sonication. Again, I have no first-hand experience, but I understand that this enzyme can be used to digest DNA until it reaches the nucleosomal core particle, and there are published ChIP-seq experiments employing this strategy. The MNase would be expected to digest unbound DNA fragments.

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If your aim is to figure out which nucleosides are specifically recognized by the protein then once you narrow down the sequence with one of the above methods, you would have to perform sequential rounds of mutagenesis.

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