Fishing DNA with Dynabeads !!

Dima Klenchin klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Mon Apr 9 11:04:14 EST 2001


"Csaba Kiss" <csakis at ki.se> wrote:
:Hi!
:I want to separate DNA spieces by their overhang
: I have to digest my DNA sample. The restriction enzyme gives a 4 bp
:overhang after digestion. 

:It is absolutely critical that I separate my
:digested DNA from the undigested. 

Why? Sounds unusual.

:I thought about using dynabeads to
:separate the digested DNA from the undigested using a 4-bp oligo hanging off
:the beads. This oligo can be biotynylated and attached to streptavidin
:beads. My main worry is if the 4-bp long hydrogen bond between the oligo and
:the digested DNA is strong enough to be able to wash away the rest of the
:DNA.

Absolutely no way it will work with 4 bp.

Here is what I can think of to achieve such specific 
purification, but it is a lot of work:

Make uneven synthetic linker that would ligate 
perfectly to your digested product, anneal it
in vitro, add to your digest, ligate, capture 
to beads carrying an oligo complementary to an 
overhang part of your linker, wash, elute by 
digestion with the same enzyme. 
Like this (requires fixed pitch font):

     
   1         2                                                    __
------- ----------------------------------------------------     /  \
--- ------------------------ ------------------------------------|  | 
                                   3                             \__/ 
                                                                4

1. your digest
2. linker
3. capturing oligo
4. insoluble support

I don't see a reason why it wouldn't work.

        - Dima




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