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Sequence specific pulldown of dsDNA


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#1 Baconman

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Posted 04 February 2015 - 08:04 AM

Hi!

Is it possible to use biotin-conjugated DNA oligos and streptavidin beads to sequence-specifically pulldown double stranded genomic DNA?

If not, are there any other ways of doing it?

 

I want to do "reverse ChIP", i.e. I want to pulldown a specific stretch of double stranded genomic DNA and then use western blot to see if my protein of interest is bound to the DNA. 

 

Cheers!

Bacon



#2 phage434

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Posted 04 February 2015 - 08:52 AM

I think this will be difficult. Chip works because you isolate protein with bound DNA. Here, you want to denature the DNA to ssDNA, hybridize the ssDNA to a probe, and isolate protein. I don't think you can denature the DNA and maintain protein crosslinking, but perhaps it would work. If the protein is bound, the DNA  may have difficulty denaturing. And if denatured, the binding to the protein may be eliminated.



#3 Baconman

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Posted 04 February 2015 - 09:31 AM

exactly, which is why I'm hoping there is a smart solution I can't think about :)



#4 mdfenko

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Posted 05 February 2015 - 05:51 AM

the way you are asking would require you to be able to make triple stranded dna.

 

maybe this paper will help:

Stable Triple-Stranded DNA Formation and its Application to the SNP Detection
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#5 Baconman

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Posted 05 February 2015 - 06:56 AM

Thanks, I will look into it.

 

Could you make a guess as to whether it would work or not? smile.png


Edited by Baconman, 05 February 2015 - 08:13 AM.


#6 mdfenko

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Posted 05 February 2015 - 12:12 PM

sorry, i have no idea if it will work.

 

it should if you can make the triple


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#7 BMF

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Posted 05 February 2015 - 01:16 PM

It is named:   DNA affinity chromatography

First paper I think is:  

 

Affinity purification of sequence-specific DNA binding proteins
 

http://www.pnas.org/...6/5889.full.pdf



#8 phage434

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Posted 05 February 2015 - 06:08 PM

This paper binds DNA of specific sequence to a column, then binds proteins to the DNA. I think this is a different probelm than what I understood here -- that the goal was to take cell lysate and isolate proteins that were already bound to specific sequences. You're right, that this may be a way to achieve a similar goal. The envirnoment will be much less like the in-vivo environment.



#9 Baconman

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Posted 05 February 2015 - 11:43 PM

Your description of the experiment I want to perform is correct phage434

Pulling down my protein with "exogenous" DNA is in my view not as good for this particular purpose, but I might give it a try anyway as a backup






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