A high-throughput protocol for deamination of long single-stranded DNA and oligo pools containing complex sequences

Yanyan Wang; Senxin Zhang; Xiaoqi Zheng; Leng-Siew Yeap; Fei-Long Meng

STAR Protocols (Dec 2023)

A high-throughput protocol for deamination of long single-stranded DNA and oligo pools containing complex sequences

Yanyan Wang,
Senxin Zhang,
Xiaoqi Zheng,
Leng-Siew Yeap,
Fei-Long Meng

Affiliations

Yanyan Wang: Shanghai Institute of Immunology, State Key Laboratory of Oncogenes and Related Genes, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
Senxin Zhang: Department of Mathematics, Shanghai Normal University, Shanghai 200234, China
Xiaoqi Zheng: Center for Single-Cell Omics, School of Public Health, Shanghai Jiao Tong University School of Medicine, Shanghai, China; Corresponding author
Leng-Siew Yeap: Shanghai Institute of Immunology, State Key Laboratory of Oncogenes and Related Genes, Department of Immunology and Microbiology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Center for Immune-Related Diseases at Shanghai Institute of Immunology, Department of Endocrinology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; Corresponding author
Fei-Long Meng: State Key Laboratory of Molecular Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Shanghai 200031, China; Corresponding author

Journal volume & issue: Vol. 4, no. 4
p. 102602

Abstract

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Summary: Cytidine deaminases as DNA mutators play important roles in immunity and genome stability. Here, we present a high-throughput protocol for deamination of long single-stranded (ss) DNA or oligo pools containing complex sequences. We describe steps for the preparation of both enzyme (activation-induced deaminase, AID) and ssDNA substrates, the deamination reaction, uracil-friendly amplification, and data analysis. This assay can be used to determine the intrinsic mutation profile of a single antibody gene or a pool of selected regions on genomic DNA.For complete details on the use and execution of this protocol, please refer to Wang et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Published in STAR Protocols

ISSN: 2666-1667 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science: Science (General)
Website: https://star-protocols.cell.com/

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