Jian Jin. William F. Kolbe and Jocelyn C. Schultz
Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Engineering Science Department, Human Genome Group, 1 Cyclotron Road, Berkeley, CA 94720, Phone: (510) 486-4082, Fax: (510) 486-5857, jian_jin@1bl.gov
It is likely that in the next generation of DNA sequencing machines, capillary array electrophoresis will be a major technology for scale-up for the Human Genome Project. In order to provide high-speed, high-throughput and cost-effective DNA sequencers to the sequencing team at LBNL's Human Genome Center and elsewhere, we have recently initiated a project to develop a practical and useful capillary array DNA sequencer. The goal of this project is to develop a fully automated DNA sequencing machine, having 96 capillary columns using a replaceable sieving matrix. The design philosophy of this machine will be to incorporate existing research results in fluorescence capillary electrophoresis studies, and implement the automation of the capillary coating, gel-filling/replacing and sample injection. To this end, we have developed a 24-capillary prototype DNA sequencing machine. The system employs the sheath-flow technique developed by N. J. Dovichi's group [1], in which an excitation laser beam traverses a gel-free flow cell and irradiates all DNA migration lanes, resulting in high-sensitivity DNA fragment detection. The fluorescence image is recorded by a cooled CCD camera. Using 6% linear polyacrylamide gel as sieving matrix, we are able to separate fragments at a speed of 500 bases/hour/capillary with an electric field of 300 V/cm, and obtain single base resolution up to 400 bases. The system is constructed in such a way that a gel-filled array of capillaries can be directly plugged into the flow cell and run immediately. After each run, the capillary array is unplugged from the cell and sent to a gel-refilling station. We have also built a sample injection apparatus which requires less than 1 µ1 of sample solution per capillary, and permits simultaneous sample injection for the whole array. Currently a 24-capillary, four color machine, which employs an image-splitting prism to separate the four colors, is being tested. The final test results will be presented.
[1] Cheng Y.F., Wu S., Chen D.Y., Dovichi N.J., Anal. Chem., 62, 496-503 (1990).
This work was supported by the Director, Office of Energy Research, Office of Health and Environmental Research, Human Genome Program, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098.