In vitro Cloning of Complex Mixtures of DNA on Microbeads: Physical Separation of Differentially Expressed cDNAs

• Published in: Proceedings of the National Academy of Sciences - PNAS 2000-02, Vol.97 (4), p.1665-1670
• Main Authors: Brenner, Sydney, Williams, Steven R., Vermaas, Eric H., Storck, Thorsten, Moon, Keith, McCollum, Christie, Jen-I Mao, Luo, Shujun, Kirchner, James J., Eletr, Sam, DuBridge, Robert B., Burcham, Timothy, Albrecht, Glenn
• Format: Article
• Language: English
• Subjects: Biological Sciences; Cloning; Cloning, Molecular - methods; Complementary DNA; Deoxyribonucleic acid; DNA; DNA Probes; DNA, Complementary - isolation & purification; Flow Cytometry; Gene expression; Gene Expression Regulation - drug effects; Genetic hybridization; Genetics; Libraries; Lipopolysaccharides - pharmacology; microbeads; Microspheres; Molecular Sequence Data; Molecules; Nucleic Acid Denaturation; Nucleic Acid Hybridization; Oligonucleotides; Polymerase Chain Reaction; Polynucleotides; RNA, Messenger - metabolism; Tetradecanoylphorbol Acetate - pharmacology; Tumor Cells, Cultured
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.97.4.1665

We describe a method for cloning nucleic acid molecules onto the surfaces of 5-μ m microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique tagging 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 ≈ 106strands complementary to one of the tags. About 105copies of each molecule are collected on each 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 bearing clones differentially expressed between two libraries by using a fluorescence-activated cell sorter (FACS). Because no prior information about the cloned molecules is required, this process is obviously useful where sequence databases are incomplete or nonexistent. More importantly, the process also permits the isolation of clones that are expressed only in given tissues or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue- or disease-directed, low-density planar microarrays.