High-Throughput Library Screening by Fluorescent Hybridizations on Gridded Membranes

Large-scale structural or functional genome analysis often deals with entire genomic or cDNA clone libraries that must be screened repeatedly with hundreds to thousands of DNA probes. High throughput protocols have been established to achieve this, making use either of massively parallel PCR screens or of hybridization experiments on high density gridded libraries. The widespread use of the latter approach has been hampered by the need to manipulate high doses of radioactivity, and to prepare good quality high density filters. We describe here a set of protocols that use “cold” fluorescent signal detection on high density filters that allows us to routinely screen about 50 probes per day on membranes each carrying 55,000 individual clones. This fast and cost-effective alternative to massive parallel PCR protocols requires little specialized equipment and can be operational within a few days.