kim@m44.unm.edu (03/06/91)
I have been thinking of methods of subtractive hybridization, with an
interest in constructing a subtracted cDNA library. One of the new methods for
separation of ds from ss DNA is to biotinylate one strand of the hybrid
("driver" DNA) to be hybridized with the other strand of the hybrid ("target").
After hybridization of the two solutions of ssDNA, all molecules bound to
biotin are isolated by using either avidin-agarose or avidin in phenol. The
subtracted target ssDNA is then extracted using heat denaturation.
I was thinking of a third way, also taking advantage of the fact that
biotinylation of the driver strand confers the ability to bind a protein
(avidin) to the driver. In this case, after hybridization, the solution would
be passed through a nitrocellulose filter. The avidin-bound molecules would
adhere to the filter, while ssTarget DNA will go through. After a couple of
washes, a hot, denaturing wash could be used to release the ss target DNA from
the hybrids.
Is this a dumb idea? What would be the capacity of a nitrocellulose filter
for avidin-biotin-DNA:DNA complexes? What is the balance between denaturing
the DNA:DNA hybrid and the Avidin:nitrocellulose or Avidin:biotin association?
Any comments? Thanks.
Daniel Kim