Eukaryotic DNA Replication: A Practical Approach (Practical Approach Series)

304 Pages·1999·13.883 MB·English

by  Sue Cotterill (Editor)

Eukaryotic DNA Replication
The Practical Approach Series
SERIES EDITOR
B. D. HAMES
Department of Biochemistry and Molecular Biology
University of Leeds, Leeds LS2 9JT, UK
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Eukaryotic DNA
Replication
A Practical Approach
Edited by
SUE COTTERILL
Marie Curie Research Institute,
Oxted, Surrey
OXPORD
UNIVERSITY PRESS
1999
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Preface
The aim of this book is to allow easy access to the techniques that are currently
in use to study eukaryotic DNA replication. The subject area that falls under
this heading is quite diverse, and its boundaries with related fields (e.g. the cell
cycle) are also rather blurred. This span is reflected in the scope of the chapters
chosen for this book which cover not only ways of working with specific repli-
cation proteins and examples of the various model systems used to study
eukaryotic DNA replication but also more general techniques. By necessity
many of the examples that are used in the text are quite specific but attempts
have been made to put these into their relevant context, and also to include
more general advice that should make the methods usable at several levels.
The field of eukaryotic DNA replication has moved fast in the last ten years
and the pace of progress shows no signs of decreasing at this point. However
the techniques that are covered here are all fundamental to the study of the
subject area. They should therefore remain useful for some time to come and
provide a good starting point for the study of any aspect of eukaryotic DNA
replication.
Oxted S.C.
May 1998
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Contents
List of con trib u tors
Abbreviations
1. Methods to map origins of replication in
eukaryotes i
Susan A. Gerbi, Anja K. Bielinsky, Chun Liang,
Victoria V. Lunyak, and Fyodor D. Urnov
1. Introduction 1
Overview 1
Methods to map origins as regions of initiation of DNA synthesis 2
Methods to map origins based on the asymmetry of bidirectional
replication 3
2. Two-dimensional (2D) and three-dimensional (3D) gels 4
Background 4
Preparation of replicating DNA 7
Neutral/Neutral (N/N) 2D gels and Southern hybridization 13
Neutral/Alkaline (N/A) 2D gels 17
3D gels 18
3. PCR analysis of nascent DNA strands 19
Background 19
DNA preparation for PCR analysis of nascent strands 21
Size fractionation of DNA 24
Purification of BUdR labelled nascent DNA 26
PCR amplification and Southern blot hybridization 28
Analysis of PCR results 30
4. Replication initiation point (RIP) mapping 31
Background 31
Labelling and isolation of DNA for RIP mapping 32
Enrichment of replication intermediate (RI) DNA on BND
cellulose 35
Phosphorylation of RI DNA by T4 polynucleotide kinase 35
\-exonuclease treatment of RI DNA 36
Primer extension reaction 37
Acknowledgements 40
References 40