Mapping replication origins by nascent DNA strand length

Methods : a Companion to Methods in Enzymology
C Staib, F Grummt

Abstract

The mapping of replication origins by nascent DNA strand length determination is a very sensitive generally applicable method that identifies even single-copy origins in mammalian chromosomes. A major advantage of this procedure is that there is no need for synchronization of cells or treatment with metabolic agents, which allows the origin to be studied under physiological conditions. This technique is based upon the amplification of specific sequence markers on nascent DNA strands that initiated replication within the region of the putative origin. Therefore, this method requires detailed sequence information of the locus to be analyzed. As a first step, nascent DNA of proliferating cells is pulse-labeled with BrdU followed by size fractionation and purification with anti-BrdU antibodies. The position of putative origins can then be determined via identification of the shortest nascent strands that can be amplified by PCR and hybridized to probes homologous to the amplified segments. Here, we give a detailed description of the theory behind the method and a full recipe for its application. Advantages and limitations of the procedure are discussed.

References

Jan 1, 1991·Annual Review of Cell Biology·W L Fangman, B J Brewer
Sep 1, 1990·Molecular and Cellular Biology·L T VassilevM L DePamphilis
Jun 16, 1989·Cell·S HandeliH Cedar
Nov 11, 1988·Nucleic Acids Research·L Vassilev, G Russev
Oct 1, 1986·Proceedings of the National Academy of Sciences of the United States of America·W C BurhansN H Heintz
Apr 1, 1984·Proceedings of the National Academy of Sciences of the United States of America·G M Church, W Gilbert
Sep 1, 1994·Molecular and Cellular Biology·E S Tasheva, D J Roufa
Dec 15, 1993·Gene·A FalaschiG Perini
Jan 1, 1993·Annual Review of Biochemistry·M L DePamphilis
Jun 1, 1993·Current Opinion in Cell Biology·M L DePamphilis
Oct 1, 1993·Molecular and Cellular Biology·V J Virta-PearlmanA C Chinault
Feb 2, 1996·The Journal of Biological Chemistry·M GenchevaG Russev
Jan 27, 1998·Methods : a Companion to Methods in Enzymology·J A Huberman

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Citations

May 2, 2006·Molecular and Cellular Biology·Rebecca K Rowntree, Jeannie T Lee
Mar 10, 1999·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·M L DePamphilis
Jan 27, 1998·Methods : a Companion to Methods in Enzymology·M L DePamphilis
Jan 27, 1998·Methods : a Companion to Methods in Enzymology·R K Clyne, T J Kelly
Jan 27, 1998·Methods : a Companion to Methods in Enzymology·J R WuD M Gilbert

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