High-Throughput Microplate Format for Producing and Screening Riboprobes from Bacterial Cells

摘要

Differential screening and subtrac-tive hybridization techniques may yield large numbers of clones enriched for differentially expressed sequences. The preferred method of selecting and confirming bona fide differentially expressed clones is to prepare each sequence as a hybridization probe and challenge Northern blots or slot blots. If the enrichment factor is modest or a low-prevalence clone is sought, screening hundreds of clones becomes desirable but is prohibitively laborious in conventional format. The need to prepare many nucleic acid probes occurs often in clinical, environmental and genomic projects. Polymerase chain reaction (PCR) based labeling protocols (2) require a microplate thermal cycler and may accommodate only 96 clones simultaneously, "Checkerboard" hybridizations (5) are limited by the size and number of nondisposable blotting manifolds. Here we present methods, which may be amenable to automation, for the growth of bacterial cultures, purification of plasmid DMA, preparation of riboprobes and nucleic acid hybridizations all in a microplate format. The simultaneous process capacity of our method is 1152 clones (12 micro-plates), the limitation imposed by centrifuge capacity and vacuum filtration manifold size.