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Updated: Oct 8 2021

Cloning

  • Overview
    https://upload.medbullets.com/topic/102099/images/20_02agenecloningpreview-l.jpg
    • Function
      • transfer production of a protein from one organism to another
      • synthesize large quantities of a plasmid or its product
    • Process
      • insertion of the vector into a bacteria via transformation
      • selection of the bacteria via a mechanism to distinguish which cells received the vector and those that did not
        • usually via an antibiotic resistance gene
    • Applications
      • recombinant proteins
        • produced by introduction of a plasmid containing the protein product of interest into bacteria
        • bacteria lysed and produced protein is purified
        • ex.) recombinant insulin, factor VIII, bacterial factors for vaccination
      • genome sequencing/libraries
        • contains the entire genome of an organism spread between bacterial colonies
        • the genome of an organism is degraded with a restriction endonuclease at specific palindromic sites
        • individual pieces are inserted into a bacterial colony and cloned individually
        • to access the library for amplification of a particular segment a blot is taken of the library and radiolabeled segment of interest is hybridized
        • the labeled colony (in which hybridization took place) can be harvested and grown
      • cDNA libraries
        • "expression libraries"
        • contain all the expressed genes of an organism in a similar fashion as a genomic library
        • requires reverse transcriptase to convert mRNA into DNA for the library
        • similar mechanism of accessing the library as the genomic library; however the probe is an 125I-antibody against the protein of interest
      • gene therapy
        • allows introduction of a normal copy of a gene into a cell that was defective in that gene
        • has been used to treat SCID caused by deficiency in the IL-receptor γ chain
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