STAR Protocols (Dec 2023)
A high-throughput protocol for deamination of long single-stranded DNA and oligo pools containing complex sequences
Abstract
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.