A major effort throughout was development of instrumentation (hardware and software) for automated, multiplexed probing of membranes. This has resulted in a number of very sophisticated machines that are able to probe and image large membranes with one probe after another so that the multiplexed DNA samples on the membrane can each be interrogated sequentially. The membrane to be probed is on the outside surface a drum that is 24 inches in diameter. This drum rotates within a second drum so that its bottom is bathed in a buffer puddle. The buffer composition is programmed to run through cycles of hybridization - washing - detection - imaging - stripping and back to the next round with the next probe. The instrument will operate unattended for 20, eight- hour cycles of hybridization to a membrane with up to a total of 1900 samples, all with automated collection of data. We discovered a novel detection method that made this process possible. Alkaline phosphatase, linked to the probe, acts on fluorogenic substrate whose product binds to the membrane maintaining the spatial localization of the probe (patent issued). The fluorescent pattern of bands is imaged while the drum rotates by excitation with a band of light and capture of the signal on a CCD. All aspects of the process are computer controlled; including temperatures, fluid deliveries, times and scan rate. In addition failsafe systems will sense problems or low fluid levels and interrupt the process without loss.
PDF
download
878987797
028-85220240
