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Updated: Jul 15, 2025

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
Published on: July 10, 2020
A high-throughput protocol for deamination of long single-stranded DNA and oligo pools containing complex sequences
Yanyan Wang1, Senxin Zhang2, Xiaoqi Zheng3
1Shanghai 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.
This study introduces a high-throughput method for DNA deamination using cytidine deaminases, like activation-induced deaminase (AID). The protocol enables detailed analysis of mutation profiles in DNA sequences, crucial for immunity and genome stability research.
Area of Science:
- Molecular Biology
- Genetics
- Immunology
Background:
- Cytidine deaminases are key DNA mutators involved in immune responses and maintaining genome stability.
- Understanding their activity is crucial for various biological processes.
Purpose of the Study:
- To develop a high-throughput protocol for DNA deamination of long single-stranded DNA or oligo pools.
- To enable detailed analysis of mutation profiles in complex DNA sequences.
Main Methods:
- The protocol involves preparing the activation-induced deaminase (AID) enzyme and single-stranded DNA (ssDNA) substrates.
- It includes steps for the deamination reaction, uracil-friendly amplification, and subsequent data analysis.
Main Results:
- The developed assay allows for the determination of intrinsic mutation profiles.
- It can be applied to single antibody genes or pools of selected genomic DNA regions.
Conclusions:
- This high-throughput deamination protocol provides a robust method for studying DNA mutator activity.
- It has significant implications for research in immunity and genome stability.
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