Elimination of Mitochondrial DNA from Mammalian Cells

Autor:	Natalya Khozhukhar, Domenico Spadafora, Yelitza A.R. Rodriguez, Mikhail Alexeyev
Rok vydání:	2018
Předmět:	0301 basic medicine Mitochondrial DNA Chemistry DNA damage Mutant General Medicine Mitochondrion Molecular biology 03 medical and health sciences chemistry.chemical_compound 030104 developmental biology Real-time polymerase chain reaction Plasmid Ethidium bromide Genotyping
Zdroj:	Current Protocols in Cell Biology. 78
ISSN:	1934-2616 1934-2500
DOI:	10.1002/cpcb.39
Popis:	To cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and new molecules are resynthesized using intact templates, if available. In this unit, we describe a method that harnesses this pathway to completely eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1). We also provide an alternate protocol for mtDNA depletion using combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC). Support protocols detail approaches for (1) genotyping ρ° cells of human, mouse, and rat origin by PCR; (2) quantitation of mtDNA by quantitative PCR (qPCR); and (3) preparation of calibrator plasmids for mtDNA quantitation. © 2018 by John Wiley & Sons, Inc.
Databáze:	OpenAIRE
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