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Development of Capillary Array DNA Sequencer for Genome Analysis
Life science has progressed greatly in the last ten years, and this progress was triggered by the human genome project. High performance genetic analyzers, especially automated fluorescent DNA sequencers, were needed for the human genome project, and several groups including Dr. Kambarafs group worked on developing applicable technologies. The developed technologies improved the DNA sequencing efficiency by two orders of magnitude over that of the conventional autoradiography method. The commercialized DNA sequencers encouraged people to start work on the human genome project, which required
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Dr. Hideki Kambara
Hitachi Corporation
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a further high performance DNA sequencer having a throughput two orders of magnitude higher than the commercialized DNA sequencers. Dr. Kambara developed a key technology for achieving the required performance. It was commercialized and later used by researchers all over the world as a principle tool for life science research, and it contributed greatly to the human genome project and to the progress in life science. Dr. Kambarafs major achievements are as follows.
1. Development of an automated DNA sequencer with side-entry laser irradiation
Although many people know the importance of the automated DNA sequencing nowadays, the importance was not recognized 30 years ago. Prof. Wada at The University of Tokyo pointed out the importance of technology for automated DNA analysis systems and started the Wada Project, which aimed to develop technologies related to DNA sequencing for the first time in the world. As one of the key project members in the project, Dr. Kambara developed a fluorescent DNA sequencer with a slab gel coupled with side-entry laser irradiation. The biggest challenge in developing a high performance fluorescent DNA sequencer was coming up with a way to achieve highly sensitive fluorescence detection and rapid electrophoresis simultaneously. He overcame the difficulty by developing a side-entry laser irradiation method where a laser irradiated all the migration tracks in a gel simultaneously from the thin gel side (0.3 mm thick), and the fluorescence image was efficiently detected with a line sensor. He also investigated in detail the characteristics of DNA separation by gel electrophoresis at various gel concentrations and found that the migration speed of DNA could increase at a low gel concentration while the DNA band separation was large enough for sequencing. At that time, he achieved the fastest real- time DNA sequencing in the world. The commercialization of fluorescent DNA sequencing technologies including Dr. Kambarafs technology triggered the initiation of the human genome project in 1990.
2. Development of capillary array DNA sequencer based on side-entry laser irradiation and sheath-flow
Two orders of magnitude higher throughput was required for the next generation DNA sequencer. To achieve the target throughput, a DNA sequencer should have as many as 96 migration tracks and should realize rapid electrophoresis with a high electric field. However, rapid electrophoresis with a high electric field produces high joulefs heat. This increases the gel temperature and destroys the gel plate and DNA bandwidths, which subsequently reduces the possible readable DNA lengths. To overcome these difficulties, gel electrophoresis with narrow capillaries was investigated by several people including Dr. Kambara. The heat radiation of a capillary system is superior to a slab gel system. However, a large electric field produced a temperature gradient in a gel capillary, where DNA fragments passing in the central region of the capillary moved faster than those passing in the outer region to produce DNA band-broadening. Consequently, capillaries with small inner diameters from 0.05-0.1 mm had to be used. Although it was preferable to have a large amount of DNA in a band for easy detection, the DNA amount as small as 1/100 of that used in the slab gel system had to be used because of a small gel tube diameter. Furthermore, the laser scanning irradiation greatly reduced the irradiation efficiency, and therefore the detection sensitivity. Although the side-entry laser irradiation was the most effective method to use in order to achieve a high detection sensitivity, it was very difficult with a capillary array system because each capillary acted as a rod lens to prevent the laser from passing through all the capillaries. This was overcome by the multiple sheath-flow technique where DNA bands eluted from the capillaries go into a buffer solution to be irradiated by the laser in a gel-free region to provide sufficient sensitivity for rapid DNA sequencing. The technique was commercialized and contributed greatly to the human genome project.
3. Fully automated capillary array DNA sequencer employing on-column laser irradiation
Although a capillary array DNA sequencer based on the sheath-flow technique achieved the target throughput, a new maintenance-free DNA sequencing system was still required because the sheath-flow cell in a capillary array sequencer needs to be cleaned frequently. Thus, a next-generation capillary array DNA sequencer was developed by employing on-column laser irradiation instead of the sheath-flow technique. Although it was very difficult, Dr. Kambara overcame the difficulties by clarifying why a laser would not go through all the capillaries and improving the production process of optical windows for capillary arrays. Capillaries are coated with polymer for reinforcement. It was necessary to make an optical window on each capillary for fluorescence detection. The windows were produced by burning out the polymer with a small burner. However, the thermal distortion on the capillaries caused twisted capillaries that could not be placed on a capillary array plane. This was overcome by developing a low temperature oxygen plasma method to remove the coatings. The developed technology was commercialized to become a world standard DNA sequencer, and it contributed to the human genome project as well as to the development of the life science field.
As mentioned above, Dr. Kambara was engaged in developing various practical technologies related to fluorescent DNA sequencers from the beginning of the genome era in the early f80s. The significant contributions of his technologies have made to genome sequencing are well known worldwide, and the human genome project would not have been completed so quickly without his technologies. Consequently, his achievements have been recognized as being worthy of a Japanese Chemical Society Award.
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