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Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling droplet array-based capillary electrophoresis and PCR-RFLP

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Abstract

The efficacy of targeted therapy is associated with multi-gene mutation status. Carrying out effective multi-genotyping analysis in combination has been a challenge in clinical settings. We therefore developed a droplet-based capillary electrophoresis (CE) system coupled with PCR-restriction fragment length polymorphism (PCR-RFLP) technology to detect multi-gene mutations from a small volume of samples. A 16 × 16 200-nL droplet array for sample encapsulation was constructed on a glass chip. The electrophoresis system consisted of a tapered vertical capillary filled with polyvinylpyrrolidone, a laser-induced fluorescence detector, and a high voltage power supply. Notably, a droplet-based electrokinetic sample introduction method and a “∩” shape capillary were developed to facilitate consecutive droplet sampling using a home-made automatic control module. The DL2000 DNA marker was consecutively separated, achieving high migration time and plate number reproducibility. The system was further applied to detect PCR-RFLP products. For colorectal cancer (CRC) cell lines, KRAS, BRAF, and PIK3CA were genotyped with a sensitivity of 0.25%. For CRC patient specimens, 30 samples were consecutively and automatically multi-genotyped without inter-sample contamination, with a lowest mutation frequency of 0.37%. For the first time, we developed a droplet-based CE system for consecutive DNA analysis with low sample consumption. This automated CE system could be further developed to integrate the full process of gene mutation detection, serving as a more effective platform for individualised therapy.

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  • 23 September 2020

    The authors would like to call the reader���s attention to the fact that unfortunately the name of Jianzhang Pan was missing as co-author of this contribution.

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Funding

This research work was financially supported by the National Natural Science Foundation of China (Grant number 81672920 and 21375149).

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Author notes

  1. Yiming Feng and Tingting Hu contributed equally to this work.

Authors and Affiliations

  1. Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, No.77 Puhe Road, Shenyang North New Area, Shenyang, 110122, Liaoning, China

    Yiming Feng, Tingting Hu, Linlin Zhou, Wanming Li & Jin Fang

  2. Institute of Microanalytical Systems, Department of Chemistry, Zhejiang University, Chemistry Experiment Building, Hangzhou, 310058, Zhejiang, China

    Pan Fang & Qun Fang

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  1. Yiming Feng
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Correspondence to Qun Fang or Jin Fang.

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The ethics committee approved the use of these samples for the purpose of academic research, and all patients who donated samples provided written informed consent.

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Feng, Y., Hu, T., Fang, P. et al. Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling droplet array-based capillary electrophoresis and PCR-RFLP. Anal Bioanal Chem 412, 3037–3049 (2020). https://doi.org/10.1007/s00216-020-02567-y

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