Identification and Isolation of Clones by Immunological Screening of cDNA Expression Libraries
Various methods are available for the construction of essentially complete cDNA or genomic libraries from many sources. Since such libraries contain thousands of members, a major difficulty is the identification of clones of interest. Final identifications are usually made by sequencing the DNA, by using the DNA to select a specific mRNA for in vitro translation, or both. Such assays are not usually suited for screening large libraries for individual clones and steps are taken to enrich for the clones of interest. The most commonly used procedures rely on cDNA hybridization probes made from an mRNA preparation that contains high levels of the sequence of interest. Variations of this general approach include the physical fractionation of the mRNA by sedimentation, gel electrophoresis, or immunoprecipitation (or absorption) of polysomes. Alternatively, if part or all of the amino acid sequence of a protein is known, it is possible to synthesize specific oligonucleotides for use either as hybridization probes themselves or as primers for the enzymatic synthesis of cDNA.
KeywordsNitrocellulose Filter cDNA Insert Expression Library Bacterial Lysate cDNA Expression Library
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