Hi,
I have never done this myself, but as far as I know, DNA can be directly
conjugated through their primary amino groups to CNBr-activated beads or
NHS-activated agarose beads. These beads are supplied by many companies:
pierce, sigma, biorad, GE healthcare, etc.. - the same thing used for making
protein-conjugated beads through amine coupling.
Unmodified DNA works, since the bases contain primary amines. In the early
days some people just loaded denatured DNA onto CNBr-activated beads and
then they would react. Here is one of the early papers:
http://onlinelibrary.wiley.com/doi/10.1111/j.1432-1033.1975.tb04151.x/pdf
My idea: if you generate sticky ends with some restriction enzymes or the
Klenow fragment, it should help exposing the bases on the overhangs, then
you should not need to denature the DNA and worry about all the crazy ways
that the DNA sits on the beads.
Having said that, ideally, terminally amine-labeled oligos should be used
whenever possible, as it is more likely to gives you higher degree of
coupling and site-specific conjugation. Only one of the two chains needs the
NH2 label, then the two complementary oligos can be annealed to make a
one-end labeled dsDNA. When a longer piece is needed, the dsDNA can be
generated by PCR with one 5'amine labeled primer and one regular primer. You
can synthesize amine labeled oligos from most oligo synthesis facilities.
Here is a literature discussing such coupling through amine modified oligos:
http://www.plosbiology.org/article/info:doi%2F10.1371%2Fjournal.pbio.0020173#pbio-0020173-g003
Amine coupling is discussed in the part "Oligonucleotide Hybridization
Chromatography".
Zhijie
--------------------------------------------------
From: "Alexandra Deaconescu" <deac...@brandeis.edu>
Sent: Saturday, April 09, 2011 8:44 PM
To: <CCP4BB@JISCMAIL.AC.UK>
Subject: [ccp4bb] immobilized DNA resin
Hello ccp4 enthusiasts:
I am afraid this is a non-ccp4 related question. Can anyone recommend an
immobilized dsDNA chromatographic resin for purification of DNA-binding
proteins? GE seems to have something - I was wondering if people have
other recommendations? In the age of GST and His tags etc., these are not
very much used, but I do not have a tag in this case...
Thanks a lot,
Alex
