| Popis: |
We have developed and are currently using a first generation fully automated hybridizer and sequence imager for multiplex sequencing. This instrument processes four multiplex membranes concurrently with a five hour cycle time, yielding a projected throughput of more than 50 million bases per year. We have also begun development of a second generation instrument which will be capable of outputs in excess of 1.5 billion raw bases per year by processing up to one hundred multiplex membranes concurrently. After direct transfer electrophoresis, each DNA containing membrane is placed in a flat tray for processing. Solutions are automatically added to and removed from the independently processed trays/membranes. In order to achieve short cycle times, a non-radioactive detection method is utilized in which oligonucleotide probes are conjugated to an enzyme and then hybridized to each of the multiplexed sequencing reaction products on the membrane. Chemiluminescent substrate is added and converted by the enzyme into an unstable product which emits light upon decay, thus revealing the sequence pattern. In the first generation instrument, the emitted light is collected at an imaging station consisting a single scientific CCD camera mounted on linear slides. The second generation instrument will increase throughput by utilizing a stationary array of cameras. By capturing segments and then tiling to form an image of the entire membrane, high collection efficiency and high resolution optics may be used to detect the extremely low light levels associated with chemiluminescence. |
