Antibody Screening of Bacteriophage λgt11 DNA Expression Libraries
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Much of our current understanding of the molecular details of the activity and interactions of proteins has stemmed from the ability to isolate cDNA encoding these proteins. Computer-based analysis of deduced amino acid sequence allows predictions to be made about their structure and function that can then be tested experimentally. The starting point for all of this is the ability to isolate the specific cDNA encoding the protein of interest from libraries that contain on the order of 105–107 recombinants. There are many approaches that can be used to isolate cDNA clones, and the exact strategy applied really depends on the molecular tools available. If a cDNA encoding the particular protein of interest has already been cloned from one species, it can be used to probe cDNA libraries from other species using DNA hybridization techniques. Alternatively, degenerate oligonucleotides can be designed from amino acid sequences from regions of proteins that are conserved across species. The oligonucleotides can be used to screen cDNA libraries by hybridization or used as primers to amplify and clone the cDNA by using the polymerase chain reaction.
KeywordsFusion Protein Nitrocellulose Filter Agar Plug Optimum Dilution Y1090 Cell
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