| 摘要 |
<p>A general vertebrate cloning and screening process for identification of genes encoding nucleic acid binding proteins is disclosed, including steps of library division and protein expression, followed by a nucleic acid binding assay as a clone screening step that is analyzed by either an Electrophoretic Mobility Shift Assay (EMSA) or a chromatography assay, preferably in a high throughput assay format. The disclosed technology provides a complete clone selection system for genes of DNA and RNA binding proteins expressed in vertebrate cells. Included are ways to optimize vertebrate cell transformation or transfection conditions by measuring the nucleic acid binding function of an expressed control protein. Also included is a high throughput electrophoretic mobility shift assay (EMSA) for detection of nucleic acid binding proteins, using a novel application of thin layer agarose gels for EMSA, combining a high vacuum and high temperature blotting technique for agarose gel desiccation with the use of a high efficiency transfer matrix (for instance, quaternary aminated nylon membranes) for blotting nucleic acid-protein complexes from agarose gels. Compensation for sample-to-sample variations in signal-to-noise ratio in clone screening is provided by introducing a reference probe to detect binding of a known protein expressed in the vertebrate host cell, along with the tester probe with the target sequence for which new clones encoding binding proteins are sought. High throughput chromatography screening methodology for nucleic acid binding proteins is also disclosed, allowing automation of screening. The disclosed technology includes combinatorial applications providing assays for functional gene linkage groups and can also be applied in connection with cDNA microarray and protein chip technologies.</p> |
