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

• Published in: Analytical and bioanalytical chemistry 2020-05, Vol.412 (13), p.3037-3049
• Main Authors: Feng, Yiming, Hu, Tingting, Fang, Pan, Zhou, Linlin, Li, Wanming, Fang, Qun, Fang, Jin
• Format: Article
• Language: English
• Subjects: Analysis; Analytical Chemistry; Arrays; Automatic control; Biochemistry; Cancer; Capillary electrophoresis; Cell Line, Tumor; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Class I Phosphatidylinositol 3-Kinases - genetics; Colorectal cancer; Colorectal carcinoma; Colorectal Neoplasms - genetics; Colorectal Neoplasms - pathology; Contamination; Deoxyribonucleic acid; Detectors; DNA; Droplets; Electric power supplies; Electrokinetics; Electrophoresis; Electrophoresis, Capillary - methods; Food Science; Gene mutations; Gene polymorphism; Genes; Genes, ras; Genetic aspects; Genetic markers; Genetic polymorphisms; Genotyping; High voltage; Humans; Laboratory Medicine; Laser induced fluorescence; Monitoring/Environmental Analysis; Mutation; Paper in Forefront; Point mutation; Polymerase chain reaction; Polymerase Chain Reaction - methods; Polymorphism; Polymorphism, Restriction Fragment Length; Polyvinylpyrrolidone; Povidone; Proto-Oncogene Proteins B-raf - genetics; Reproducibility of Results; Restriction fragment length polymorphism; Tumor cell lines
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-020-02567-y

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.