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Abstract (1. First Research Institute of the Ministry of Public Security of PRC, Beijing 100048, China; 2. Institute of Forensic Science of China, Beijing 100038, China; 3. Bio-evidence Sciences Academy, Xi’an Jiaotong University & National Biosafety Evidence Foundation (NBEF), Xi’an 710049, China; 4. Beijing Zhongdun Anmin Analysis Technology CO. Ltd., Beijing 102200, China)
Abstract: A domestic 24 channels genetic analyzer combined with a domestic 36 cm capillary arrays were used to solve the problems of fluorescence signal correction, running voltage, and migration correction during sequencing on the genetic analyzer based on non-gel sieving capillary electrophoresis technology. Spectral calibration model and appropriate baseline noise values were obtained under the condition that the original spectral fluorescence signal of the analyzer was used as a regulation basis. A standard curve was plotted by combining an running voltage and a base spacing value, while a clear range length was also calculated by a sequencing analysis software, so as to determine the optimal running voltage and base spacing. In addition, a migration offset value was further calculated and a linear model was eventually established between the base size and the migration time, which achieved an accurate identification of bases. It was reported that with the appropriate spectral calibration model and baseline noise threshold limitation, the strongest ability to detect the longest clear range length, that is 561 bp, was found in the analyzer when the running voltage was set to 10 kV and the base spacing was 13.05 frames. By compensating for migration corrections, a linear model was established between the base size and the migration time. Among them, R2 values of bases G, A, T, and C increased from 0.992 7, 0.992 7, 0.994 5, and 0.987 9 to 0.999 6, 0.999 8, 0.999 6, and 0.999 7, respectively. After correction, all fluorescent bases were accurately labeled without any omissions or errors, clear range length extended to 621 bp. This study could better guide the optimization and design of a sequencing module of the domestic genetic analyzer, making the DNA base recognition function of the analyzer more efficient and accurate.
Key words: domestic genetic analyzer; non-gel capillary electrophoresis; base sequencing; run voltage; migration correction
（Acta Laser Biology Sinica, 2024, 33(3): 217-226）
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YANG Yue, WANG Hui, WANG Rui, MA Ke, XU Ying, ZHANG Shuijun, ZHANG Zhenzhen, CHEN Rong, MENG Qingguo, FENG Zhixin. Expression, Purification, and Preliminary Crystallographic Study of Ferritin from Spiroplasma eriocheiris#br#[J]. journal1, 2024, 33(3): 227-235. |
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