Purification of Restriction Fragments Containing Replication Intermediates from Complex Genomes for 2-D Gel Analysis

  • Larry D. Mesner
  • Pieter A. Dijkwel
  • Joyce L. Hamlin
Part of the Methods in Molecular Biology book series (MIMB, volume 1300)


In order to perform 2-D gel analyses on restriction fragments from higher eukaryotic genomes, it is necessary to remove most of the linear, nonreplicating, fragments from the starting DNA preparation. This is so because the replication intermediates in a single-copy locus constitute such a minute fraction of all of the restriction fragments in a standard DNA preparation—whether isolated from synchronized or asynchronous cultures. Furthermore, the very long DNA strands that characterize higher eukaryotic genomes are inordinately subject to branch migration and shear. We have developed a method that results in significant enrichment of replicating fragments that largely maintain their branched intermediates. The method depends upon two important factors: (1) replicating fragments in higher eukaryotic nuclei appear to be attached to the nuclear matrix in a supercoiled fashion, and (2) partially single-stranded fragments (e.g., those containing replication forks) are selectively adsorbed to benzoylated naphthoylated DEAE (BND)-cellulose in high salt concentrations. By combining matrix-enrichment and BND-cellulose chromatography, it is possible to obtain preparations that are enriched as much as 200-fold over the starting genomic DNA, and are thus suitable for analysis on 2-D gels.

Key words

Replication intermediates Nuclear matrix BND-cellulose chromatography 2-D gel replicon mapping 



We thank the present and former members of our laboratory for very helpful discussions. This work was supported by a grant from the NIH to J.L.H. and L.D.M. (RO1 GM26108).


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Larry D. Mesner
    • 1
  • Pieter A. Dijkwel
    • 2
  • Joyce L. Hamlin
    • 2
  1. 1.Department of Public Health Sciences, Health Science CenterUniversity of Virginia School of MedicineCharlottesvilleUSA
  2. 2.Department of Biochemistry and Molecular Genetics, Health Science CenterUniversity of Virginia School of MedicineCharlottesvilleUSA

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