In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs

We describe a method for cloning nucleic acid molecules onto the surfaces o f 5-mu m microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique t agging of the molecules is achieved by sampling a small fraction (1%) of a very large repertoire of tag sequences. The resulting library is hybridized to microbeads that each carry approximate to 10(6) strands complementary t o one of the tags. About 10(5) copies of each molecule are collected on eac h microbead, Because such clones are segregated on microbeads, they can be operated on simultaneously and then assayed separately. To demonstrate the utility of this approach, we show how to label and extract microbeads beari ng clones differentially expressed between two libraries by using a fluores cence-activated cell sorter (FACS), Because no prior information about the cloned molecules is required, this process is obviously useful where sequen ce databases are incomplete or nonexistent. More importantly, the process a lso permits the isolation of clones that are expressed only in given tissue s or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue- or dis ease-directed, low-density planar microarrays.