Saika Aytay, Maureen Thornton[1], Lisa Davis, Charles Helmstetter[1], and John Hozier
Applied Genetics Laboratories, Melbourne, Florida, 32901.
MICROGENETICS[TM] is a new method for screening large insert libraries with current emphasis on screening yeast artificial chromosome (YAC) libraries. The combination of techniques under development allows library screening at the microscopic level. We take advantage of a well-established culturing technique, the "baby machine", in which a population of dividing "mother" cells is attached firmly to a surface. In MICROGENETICS the progeny cells are collected as microclones on a parallel surface in a pattern identical to the original, thus forming a replica. Cells on the replica surface are fixed in-situand sequence-specific probes are hybridized to corresponding YAC DNAs using standard FISH techniques. Alternatively clones of interest are identified by in-situ PCR. Positive microclones are detected by fluorescence microscopy and the viable positive mother cell is removed from the mother plate with a micromanipulator-driven micropipette, and cultured and propagated for further analysis. With MICROGENETICS a YAC library of 30,000 clones can be maintained and screened on a single microscope slide providing a many-fold increase in efficiency over current technology.
During the process of developing this technology we have used several procedures to attach the yeast cells to glass slides, since normally yeast cells do not attach to glass. We have coated slides with poly-lysine, Cell-Tak, silane and Con-A. Individual colonies remain attached to Con-A treated slides during the process of in-situ PCR and fluorescence detection if the cells are fixed with methanol and acetic acid after replica formation. We have performed in-situ PCR on replica colonies using several sets of primers with unique sequences which target specific dinucleotide repeat markers in known mouse YAC clones which were characterized in earlier studies. With in-situ PCR we obtained the same patterns of positive and negative results with respect to markers used for each YAC clone as predicted by our previous studies. We have also spiked YAC clones known to be negative for a given primer set with positive YAC clones in known ratios and proceeded with in-situ PCR and fluorescence detection, and observed the expected ratio of positive to negative signals. Similar experiments are being performed to detect positive colonies at lower frequencies which will mimic positive colonies in standard YAC libraries. We are also forming gridded arrays of known positives in a background of negatives. Lastly, we will isolate mother cell microcolonies for propagation and show that they are indeed the positive colonies which we spiked among negative colonies.
MICROGENETICS technology can be applied readily to other large insert libraries with bacterial hosts (BAC's, P1's), since the "baby machine" technique was originally designed for E. coli. This technology reduces a given library from a stack of hundreds of microtiter plates to a single surface and gives a clone individualized treatment only after identification of the clone of interest in the screening step.
[1] Department of Biology, Florida Institute of Technology, Melbourne, Florida 32901.
*Supported by grant DE-FG-02-95ER 81924 from the Director, Office of Health and Environmental Research, Health Effects and Life Sciences Research Division of the U.S. Department of Energy.