Combined bidimensional electrophoresis and electron microscopy to study specific plasmid DNA replication intermediates in human cells
| Autor: | Cindy Follonier, Massimo Lopes |
|---|---|
| Přispěvatelé: | University of Zurich, Stockert, Juan C, Espada, Jesús, Blázquez-Castro, Alfonso |
| Jazyk: | angličtina |
| Rok vydání: | 2014 |
| Předmět: |
10061 Institute of Molecular Cancer Research
DNA replication Biology Origin of replication Molecular biology Virus 3. Good health Cell biology chemistry.chemical_compound genomic DNA Plasmid chemistry 1311 Genetics In vivo Replication (statistics) 1312 Molecular Biology Agarose 570 Life sciences biology |
| Zdroj: | Methods in Molecular Biology ISBN: 9781627037051 |
| DOI: | 10.5167/uzh-83872 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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