How to covalently link DNA to beads?
malexeyev at biost1.thi.tmc.edu
Wed Feb 21 14:28:50 EST 1996
In article <lamphier-210296003535 at pl-urawa035.infosphere.or.jp>,
lamphier at po.infosphere.or.jp (Marc Lamphier) wrote:
> Does anyone know where I can find methods for covalently linking DNA
> (lambda phage) to beads of some sort (Sepharose, etc.). The beads should be
> able to be sorted through a FACS cell sorter. I am trying to develop a
> method of using fluorescent probes to screen a lambda library immobilized
> on beads via FACS.
> Marc Lamphier
> University of Tokyo
I think you can use streptavidin-agarose gel from many sources and lambda
DNA labelled with biotin (say, using terminal transferase and biotinylated
dNTP). Avidin-biotin interaction is irreversible for most practical
purposes, I think.
On the other hand, you can perform mild periodate oxidation of your
agarose derivative (sepharose) and introduce chemically reactive groups
(i.e. sulfhydryl) that can be later used to covalently link DNA through
5' end. 5' PO4 group on DNA can be modified to be SH- or NH2- and later
used for coupling to a support. For example, you can use PDPH to
introduce pyridyldithio group in your matrix (agarose-based), modify 5'
on DNA with cystamine, reduce it with DTT to provide free SH-group and mix
with modified matrix. Cleavable covalent S-S bond will be formed between
matrix and DNA. If noncleavable bond is needed you may use M2C2H or MPBH
to modify matrix instead of PDPH. Alternatively, you may reduce
PDPH-derived matrix with DTT prior DNA coupling and perform coupling in
the presence of BMH.
If you need more info, contact me or call people in Pierce (no connections
other than customer).
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