Presentation on theme: "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."— Presentation transcript:
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)
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)
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.
Caffeine Elution Benzoyl Chloride2-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.
2D Replication Structures DNA digested with restriction enzymes 1 st dimension separates by MASS 2 nd 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
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
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
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
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