bnd cellulose useful for isolation of dna and rna from crude lysates can also be used to separate...

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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)

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Page 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

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)

Page 2: 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

BND Cellulose

“RCIs can be enriched by BND-cellulose chromatographyTo 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)

Page 3: 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

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 Cellulose…perhaps it’s because they don’t know and just use what has worked since the 1960’s.

Page 4: 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

Caffeine Elution

Benzoyl Chloride 2-Naphthol

Apparently no one in DNA Replication Intermediate Enrichment Research wants to let a person like me know why caffeine elutes ssDNA off of BND Cellulose…perhaps it’s because they don’t know and just used what has worked since the 1960’s.

Mahta’s Caffeine Explanation

Caffeine

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.

Page 5: 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

2D Replication Structures

Dijkwel et. al., MCB 1991

Page 6: 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

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

Page 7: 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

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

Page 8: 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

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

Page 9: 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

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

Page 10: 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

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