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<p>Capillary electrophoresis (CE) plays an important role in the quality control of dsDNA. So far, there has been various fluorescent dyes employed for the separation of dsDNA by CE. However, the molecular weight of the dyes may affect the mass to charge ratio of dsDNA-dye complex, consequently the separation performance of dsDNA will be changed. Herein, we systematically compared the fluorescent intensity and migration times when separating the dsDNA fragments labeled or intercalated by different dyes. Results showed that the concentration of SYBR Green I affected the migration times more than Gel Green and EvaGreen, which may be caused by the lower molecular weight of EvaGreen. The optimal concentration for SYBR Green I and Gel Green is 1×, and it is 0.005× for EvaGreen. There is linear relationship between dsDNA concentration (0.1–0.5 ng/µL) and fluorescence intensity when using SYBR Green I or Gel Green for separation. Finally, we have resolved the фX174-Hinc II digest in 0.5% HEC (1300k) containing 1× SYBR within 12&#xa0;min, even though there is only 6&#xa0;bp difference for the adjacent dsDNA fragments. Furthermore, we also obtained the virtual dsDNA bands by OpenCV according to the electropherogram.</p>

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Effect of Molecular Weight of Fluorescent Dyes on DNA Separation by Capillary Electrophoresis

  • 期刊论文

  • 发表日期：

    2025年3月13日

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  • Yuqing Xu,
  • Xin Chen,
  • Shuaiqiang Fan,
  • Ting Zhang,
  • Bo Yang,
  • Zhenqing Li,
  • Shintaro Yamaguchi,
  • Dawei Zhang

摘要

Capillary electrophoresis (CE) plays an important role in the quality control of dsDNA. So far, there has been various fluorescent dyes employed for the separation of dsDNA by CE. However, the molecular weight of the dyes may affect the mass to charge ratio of dsDNA-dye complex, consequently the separation performance of dsDNA will be changed. Herein, we systematically compared the fluorescent intensity and migration times when separating the dsDNA fragments labeled or intercalated by different dyes. Results showed that the concentration of SYBR Green I affected the migration times more than Gel Green and EvaGreen, which may be caused by the lower molecular weight of EvaGreen. The optimal concentration for SYBR Green I and Gel Green is 1×, and it is 0.005× for EvaGreen. There is linear relationship between dsDNA concentration (0.1–0.5 ng/µL) and fluorescence intensity when using SYBR Green I or Gel Green for separation. Finally, we have resolved the фX174-Hinc II digest in 0.5% HEC (1300k) containing 1× SYBR within 12 min, even though there is only 6 bp difference for the adjacent dsDNA fragments. Furthermore, we also obtained the virtual dsDNA bands by OpenCV according to the electropherogram.