In Situ Analysis of Mitochondrial DNA Synthesis Using Metabolic Labeling Coupled to Fluorescence Microscopy.

Autor:	Smolka JA; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA., Lewis SC; Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA. samlewis@berkeley.edu.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2023; Vol. 2615, pp. 99-106.
DOI:	10.1007/978-1-0716-2922-2_8
Abstrakt:	Metabolic labeling with the nucleoside analog 5-ethynyl-2'-deoxyuridine (EdU) enables the selective labeling of DNA synthesis in live cells. Newly synthesized EdU-containing DNA can be covalently modified after extraction or in fixed cells using copper-catalyzed azide-alkyne cycloaddition "click chemistry" reactions, enabling bioconjugation to various substrates including fluorophores for imaging studies. While often used to study nuclear DNA replication, EdU labeling can also be leveraged to detect the synthesis of organellar DNA in the cytoplasm of Eukaryotic cells. In this chapter, we outline methods for the application of EdU labeling to the study of mitochondrial genome synthesis in fixed cultured human cells, using fluorescent labeling and superresolution light microscopy. (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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