Consecutive and automatic detection of multi-gene mutations from colorectal cancer samples by coupling droplet array-bas
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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 Yiming Feng 1 & Tingting Hu 1 & Pan Fang 2 & Linlin Zhou 1 & Wanming Li 1 & Qun Fang 2 & Jin Fang 1 Received: 22 January 2020 / Revised: 20 February 2020 / Accepted: 2 March 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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. Keywords Capillary electrophoresis . Droplets . Multi-gene detection . Gene mutation . PCR-RFLP
Introduction Gene mutation is a critical molecular change occurring during cancer. Notably, it was found that gene mutation is related to the efficacy of cancer treatments such as targeted therapy. Although the introduction of targeted therapy has shown superior specificity and efficiency to
traditional chemoradiotherapy, only a portion of patients can benefit from this due to their differentiating genotypes [1, 2]. For example, the KRAS mutation was identified as a negative biomarker for anti-EGFR-targeted therapy [3]. Santos et al. [4] reported that the KRAS mutation is associated with a worse outcome for anti-EGFR-targeted therapy compared with wild-type KRAS. The response rates
Yiming Feng and Tingting Hu contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00216-020-02567-y) contains supplementary material, which is available to authori
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