Citation: Huige Zhang, Wei Chen, Yuyan Huang, Mingfang Wu, Hongli Chen, Cuiling Ren, Xiaoyan Liu, Haixia Zhang. Construction of template-free amplification system coupled with capillary electrophoresis for the simultaneous detection of three tumor-associated DNA repair enzymes[J]. Chinese Chemical Letters, ;2025, 36(9): 110721. doi: 10.1016/j.cclet.2024.110721 shu

Construction of template-free amplification system coupled with capillary electrophoresis for the simultaneous detection of three tumor-associated DNA repair enzymes

College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China

zhanghx@lzu.edu.cn

Received Date: 14 June 2024
Revised Date: 20 October 2024
Accepted Date: 3 December 2024
Available Online: 4 December 2024

Figures(6)

DNA repair enzymes are important in the repair of DNA lesions for maintaining the genome stability, and their abnormal expression induced various human cancers. Simultaneous detection of these DNA enzymes could provide convincing evidence based on the comparison of the activity of multiple enzymes than on that of single enzyme. Although fluorescence approach has been applied for the simultaneous detection both of DNA repair enzymes, the spectral overlap and multiwavelength excitation severely restrict the number of available fluorophores. Thus, it is difficult to simultaneously detect three enzymes in a single analysis by fluorescence detection. Herein, we developed a method for the simultaneous determination of three DNA repair enzymes including human flap DNA endonuclease 1 (FEN1), human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG) based on the combination of template-free amplification system with capillary electrophoresis-laser induced fluorescence (CE-LIF) detection. The amplification system was adopted to transfer and amplify the enzymatic products into different length DNA fragments which could be separated effectively by CE-LIF without the complicated modification of the capillary inner wall or labeling different tails on signal probes for separation. The method demonstrated a detection limit of 0.07 U/mL (0.08-160 U/mL) for FEN1, 2.40 U/mL (2.5-250 U/mL) for hAAG and 2.1 × 10⁻⁴ U/mL (0.0004-2.5 U/mL) for UDG, the relative standard deviations (RSDs) of peak time and peak area for different analytes were as follows: 2.50%-4.37% and 3.24%-7.18% (inter-day); 1.37%-2.71% and 1.43%-3.02% (intra-day), 4.28%-6.08% and 4.16%-7.57% (column to column), respectively. And it can identify the inhibitor-like drugs, evaluate enzymatic kinetics and achieve the detection of three enzymes in cell extracts, providing a simple and powerful platform for simultaneous detection of more DNA repair enzymes.
- Simultaneous detection,
- Template-free amplification,
- DNA repair enzymes,
- Capillary electrophoresis
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