| DNA sequence analysis plays an important role in modern biology and life science. DNA analysis apparatus is the key scientific instrument for DNA sequence analysis, which can be widely used in medical, agricultural and forensic fields. Studies on DNA sequence analysis and its correlative technologies are not only a scientific branch, but also a high technology industry, which covers multi-disciplines such as microfabrication, polymer science, analytical chemistry, biological engineering, optical engineering and so on. Correlative technologies and applications in DNA analysis have developed greatly in China. Some research results have reached or have been closed to international top level. But in some key technologies such as high speed, high throughput and high sensitivity detecting, our researches still have a large gap with the international top level. This dissertation carries on the research on fluorescence signal acquisition and processing system in DNA analysis instrument, which has high performance and independent intellectual property right:1. Scanning mirror and self designed large numerical aperture (N.A.) telecentric f-theta objective were adopted to realize parallel detection of multi-channel DNA fluorescence signals. And its suitable control system was also designed to achieve low noise, high sensitivity confocal scanning detection.2. As referring to the optical part of the signal acquisition system, theoretically, the diffraction of Gaussian beam limited by aperture and the line resolution of laser beam scanning systems were analyzed in detail. The diffraction distributions of Gaussian beam limited by the circular apertures with different sizes in scanning objective's focal plane were studied, and then the effective spot radius in focal plane was calculated using second moment and compared with the ideal one. The influence of apertures with different sizes on resolution was analyzed. The results show that diffraction limited by the small apertures will lower the system's resolution. But with the increasing of aperture's radius, the influence becomes smaller, when the radius l≥2ω, the influence can be ignored. These results are helpful for the design of confocal scanning detection system, and may be helpful for laser beam's application in all optical communications, integrated optics and so on.3. According to its flow chart in DNA analysis system, the signals processing were studied systematically, including pre-processing (data set selection, baseline adjustment, denoising, peak identification); four-color fluorescence signals crosstalk correction; post-processing (deconvolution, mobility shifting correction, signals intensity normalization). As referring to the research of optimizing of denoising method, in order to construct the same noise model as that in experiment and evaluate the denoising algorithm precisely, a novel method was presented: real noise data acquired from the experimental system were added to an ideal signal to simulate a noisy DNA sequencing signal, thus the denoising efficiency could be evaluated accurately. The denoising results indicate that sym7 is the optimal wavelet base, it can effectively reduce the noise of DNA sequencing signal. After processed, the relative errors of the signals' peak height and peak position are smallest, the SNR is the highest, and the RMSE is also the smallest, comparison to that of db8, coif5 etc.Based on the above theoretical researches and innovation design, this dissertation finished the research and development of the DNA analysis instrument's fluorescence signal acquisition and processing system. The fast parallel detection of multi-channel DNA fluorescence signal was realized by a telecentric f-theta objective and a scanning mirror; the four-color fluorescence signal's confocal detection was realized by a filter set and a photomultiplier tube (PMT). The signal acquisition apparatus integrated an embedded system, the chromatograms could be displayed in a live mode, and the data could be stored and processed in a computer..Novel designed DNA fluorescence signal acquisition system only uses one PMT, which lowers the manufacture cost of the system; the system's working noise by using optical scanning method is much lower than that by mechanical scanning, and the detecting sensitivity is high as well. Capillary electrophoresis experiments were executed in the system using DNA marker pBR322/Hae III. The system's limit of detection was evaluated to be 2.368×10-12 mol/L (dsDNA with Thiazole Orange). Cooperating with the chromatogram processing software, the system can meet the requirement of single base pair resolution in DNA analysis. The system is expected to be applied to both capillary array and multi-channel microchip electrophoresis detection based on laser-induced fluorescence.
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