BND Cellulose

1
BND Cellulose

• Useful for isolation of DNA and RNA from crude
  lysates
• Can also be used to separate ssDNA from dsDNA
• dsDNA comes off by gravity flow in 800mM NaCl,
  10mM Tris pH 8.0, 1mM EDTA
• ssDNA is generally eluted off with a gradient of
  0 to 2 caffeine in 1.0M NaCl, 10mM Tris pH 8.0,
  1mM EDTA
• More than 95 of Replication Intermediates (RIs)
  partition with the caffeine elution (Dijkwel et.
  al., MCB 1991)
• Adding 50 formamide in a subsequent step elutes
  more RIs (Feng et. al., Genetics
  1995)

2
BND Cellulose
RCIs can be enriched by BND-cellulose
chromatography To verify the presence of
replication forks on the putative RCIs that we
detected on 2D gels, we asked whether the
molecules that comprise the sigmoid arcs could be
enriched by BND-cellulose chromatography. Such
enrichment is used for the analysis of
replication intermediates in many eukaryotic
systems. It is based on the binding of the
single-stranded region that exists in any
replication fork to the column, and its
separation from the majority of the
double-stranded non-replicating DNA. The
replication forks can be released from the column
by caffeine. (Cohen et. al., Nucleic Acids
Research 2005)
3
Caffeine Elution
Apparently no one in DNA Replication Intermediate
Enrichment Research wants to let a person like me
know why caffeine elutes ssDNA off of BND
Celluloseperhaps its because they dont know
and just use what has worked since the 1960s.
4
Caffeine Elution
Apparently no one in DNA Replication Intermediate
Enrichment Research wants to let a person like me
know why caffeine elutes ssDNA off of BND
Celluloseperhaps its because they dont know
and just used what has worked since the 1960s.
Mahtas Caffeine Explanation
Benzoyl Chloride
2-Naphthol
Benzoylated Naphthol has a lower affinity for
dsDNA than it does for ssDNA it comes off with
a high salt wash. Caffeine is able to compete
off the ssDNA from the resin.
Caffeine
5
2D Replication Structures
Dijkwel et. al., MCB 1991
6
2D Replication Structures

• DNA digested with restriction enzymes
• 1st dimension separates by MASS
• 2nd dimension separates by STRUCTURE and MASS (it
  is a higher gel the shape contributes a lot
  to the migration rate)
• Non-replicating DNA migrates as a single band
• Replicating fragments migrate at slower rate
  according to extent it has been replicated
• Typical RIs separate by 2D neutral/neutral gel
  electrophoresis
• Hybridization with probes for fragments that
  contain different elements

7
2D RIs Simple Y or Fork Arc

• Range of Y molecules
• (b) results from a fragment that is replicated
  passively from an outside origin
• (a) nonreplicating fragments from the genome as
  a whole
• Based on a computational approach as to where
  these structures are predicted to reside???

Dijkwel et. al., MCB 1991
8
2D RIs Centered Origin of Replication

• (c) Pattern from a fragment with a centered
  origin of replication probed
• bubbles migrate slower at all extents of
  replication than do forks in a fragment of equal
  mass

Dijkwel et. al., MCB 1991
9
2D RIs Off-centered Origin

• (c) Incomplete bubble arc arises from an
  off-centered origin in a fragment
• This reverts to (b) or fork arc when the bubble
  expands beyond the right-hand restriction site
  fork breaks

Dijkwel et. al., MCB 1991
10
2D RIs Approaching Forks

• (d) or (e) arises when two forks approach each
  other in a fragment symmetrically (d) or
  asymmetrically (e)
• (f) if there is a fixed terminus in a fragment,
  the collected X-shaped structures would result in
  a concentrated spot on this curve
• The triangle between (b) simple Y arc, (e) and
  (f) contains a collection of double-forked
  structures differing in extents of replication
  and positions of the fork within the fragment

Dijkwel et. al., MCB 1991