Enrichment of plasmid pool from E. coli for mass sequencing to detect untargeted mutagenic events

Asako Isogawa; Robert P Fuchs; Shingo Fujii

doi:10.1016/j.xpro.2021.100399

Enrichment of plasmid pool from E. coli for mass sequencing to detect untargeted mutagenic events

STAR Protoc. 2021 Mar 20;2(2):100399. doi: 10.1016/j.xpro.2021.100399. eCollection 2021 Jun 18.

Authors

Asako Isogawa^{1

2

3

4}, Robert P Fuchs^{4

5

6}, Shingo Fujii^{1

2

3

4}

Affiliations

¹ CNRS, UMR7258, Marseille 13009, France.
² Inserm, U1068, Marseille 13009, France.
³ Institut Paoli-Calmettes, Marseille 13009, France.
⁴ Aix-Marseille University, UM 105, Marseille 13284, France.
⁵ Marseille Medical Genetics, UMR1251, Marseille 13385, France.
⁶ Inserm, U1251, Marseille 13385, France.

Abstract

Translesion synthesis (TLS) is an event to cope with DNA damages. During TLS, the responsible TLS polymerase frequently elicits untargeted mutagenesis as potentially a source of genetic diversity. Identifying such untargeted mutations in vivo is challenging due to the bulk of DNA that does not undergo TLS. Here, we present a protocol to enrich a plasmid pool that underwent Pol V-mediated TLS in Escherichia coli for mass sequencing. The concept of this protocol could be applied into any species. For complete details on the use and execution of this protocol, please refer to Isogawa et al. (2018).

Keywords: Genetics; Model Organisms; Molecular Biology.

MeSH terms

DNA Damage / genetics
DNA Mutational Analysis / methods*
DNA, Bacterial / genetics*
Escherichia coli / genetics*
Mutagenesis / genetics
Mutation / genetics*
Plasmids* / genetics
Plasmids* / isolation & purification
Plasmids* / metabolism

Substances

DNA, Bacterial