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Combined bidimensional electrophoresis and electron microscopy to study specific plasmid DNA replication intermediates in human cells


Follonier, Cindy; Lopes, Massimo (2014). Combined bidimensional electrophoresis and electron microscopy to study specific plasmid DNA replication intermediates in human cells. In: Stockert, Juan C; Espada, Jesús; Blázquez-Castro, Alfonso. Functional Analysis of DNA and Chromatin. New York, USA: Humana Press (Springer), 209-219.

Abstract

Replication interference by specific chromosomal sequences—such as trinucleotide repeats—plays a causative, though undefined role in the aetiology of human disease, especially neurodegenerative syndromes. However, studies on these mechanisms in human cells have been hampered by poorly defined replication origins on genomic DNA. Simian Virus 40 (SV40)-based plasmids were useful in the past to overcome these experimental limits, but have been rarely amenable for the most complex and revealing molecular biology approaches to study in vivo DNA replication interference. This chapter describes a new, safe, SV40-based episomal system that replicates with very high efficiency in human cells and allows isola- tion of in vivo replication intermediates with high yield and purity. We describe how to use this experimen- tal system to run preparative agarose 2D-gel and to extract specific replication intermediates to visualize by electron microscopy.

Abstract

Replication interference by specific chromosomal sequences—such as trinucleotide repeats—plays a causative, though undefined role in the aetiology of human disease, especially neurodegenerative syndromes. However, studies on these mechanisms in human cells have been hampered by poorly defined replication origins on genomic DNA. Simian Virus 40 (SV40)-based plasmids were useful in the past to overcome these experimental limits, but have been rarely amenable for the most complex and revealing molecular biology approaches to study in vivo DNA replication interference. This chapter describes a new, safe, SV40-based episomal system that replicates with very high efficiency in human cells and allows isola- tion of in vivo replication intermediates with high yield and purity. We describe how to use this experimen- tal system to run preparative agarose 2D-gel and to extract specific replication intermediates to visualize by electron microscopy.

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Additional indexing

Item Type:Book Section, not refereed, original work
Communities & Collections:04 Faculty of Medicine > Institute of Molecular Cancer Research
07 Faculty of Science > Institute of Molecular Cancer Research
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2014
Deposited On:04 Nov 2013 08:00
Last Modified:05 Apr 2016 17:05
Publisher:Humana Press (Springer)
Series Name:Methods in Molecular Biology
Number:1094
ISSN:1064-3745
ISBN:978-1-62703-705-1
Publisher DOI:https://doi.org/10.1007/978-1-62703-706-8_16
Related URLs:http://link.springer.com/book/10.1007/978-1-62703-706-8
PubMed ID:24162990

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