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DNA replication mediated by DNA polymerases is important not only for replicating genomic DNA but also for repairing damaged DNA. Elucidating the function of each DNA polymerase is essential for understanding how genomic integrity is maintained. Here, we present two types of DNA polymerase assays: one is the conventional method using sequence gel and the other is the single-molecule Förster resonance energy transfer (smFRET) measurement. The conventional method is sensitive and suitable for detecting the end product of DNA replication. On the other hand, the smFRET assay can monitor the intermediate stages of the reaction such as the synapsis between two single-stranded DNAs (ssDNAs). The two assays complement each other and provide information for more detailed analysis when used together.

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Measurement of DNA Polymerase and Microhomology-Mediated End-Joining Activities by Gel Electrophoresis and smFRET Techniques

  • 实验方案

  • 发表日期：

    2025年

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  • Yubin Sung,
  • Chanwoo Kim,
  • Hajin Kim,
  • Kei-ichi Takata

摘要

DNA replication mediated by DNA polymerases is important not only for replicating genomic DNA but also for repairing damaged DNA. Elucidating the function of each DNA polymerase is essential for understanding how genomic integrity is maintained. Here, we present two types of DNA polymerase assays: one is the conventional method using sequence gel and the other is the single-molecule Förster resonance energy transfer (smFRET) measurement. The conventional method is sensitive and suitable for detecting the end product of DNA replication. On the other hand, the smFRET assay can monitor the intermediate stages of the reaction such as the synapsis between two single-stranded DNAs (ssDNAs). The two assays complement each other and provide information for more detailed analysis when used together.