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Single Cell Analysis of Mitochondrial DNA Deletions

Lookup NU author(s): Dr Helen Tuppen, Dr Amy Reeve, Dr Amy VincentORCiD


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© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.Mitochondrial DNA (mtDNA) deletions underpin mitochondrial dysfunction in human tissues in aging and disease. The multicopy nature of the mitochondrial genome means these mtDNA deletions can occur in varying mutation loads. At low levels, these deletions have no impact, but once the proportion of molecules harbouring a deletion exceeds a threshold level, then dysfunction occurs. The location of the breakpoints and the size of the deletion impact upon the mutation threshold required to cause deficiency of an oxidative phosphorylation complex, and this varies for each of the different complexes. Furthermore, mutation load and deletion species can vary between adjacent cells in a tissue, with a mosaic pattern of mitochondrial dysfunction observed. As such, it is often important for understanding human aging and disease to be able to characterise the mutation load, breakpoints and size of deletion(s) from a single human cell. Here, we detail protocols for laser micro-dissection and single cell lysis from tissues, and the subsequent analysis of deletion size, breakpoints and mutation load using long-range PCR, mtDNA sequencing and real-time PCR, respectively.

Publication metadata

Author(s): Tuppen HAL, Reeve AK, Vincent AE

Publication type: Book Chapter

Publication status: Published

Book Title: Mitochondrial DNA : Methods and Protocols

Year: 2023

Volume: 2615

Pages: 443-463

Print publication date: 27/03/2023

Online publication date: 21/02/2023

Acceptance date: 02/04/2022

Series Title: Methods in Molecular Biology

Publisher: Humana Press, Inc.

Place Published: New York


DOI: 10.1007/978-1-0716-2922-2_29

PubMed id: 36807808

Notes: 9781071629222 ebook ISBN

Library holdings: Search Newcastle University Library for this item

ISBN: 9781071629215