Updated: 10/8/2021

Cloning

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  • Overview
    • Function
      • transfer production of a protein from one organism to another
      • synthesize large quantities of a plasmid or its product
    • Process
      • insertion of a DNA segment of interest into a vector
        • vector is usually a plasmid
          • first: digestion of vector with restriction endonuclease
          • second: sealing of the segment into the vector with DNA ligase
      • 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
        • bacterial colony grown
          • some plasmids contain regulatory sequences which can turn on/off expression of the plasmid
        • 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
        • because mRNA has had the non-coding introns spliced out it can express genes from the cloned host
          • bacteria do not contain the enzymes to splice RNA
        • 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
        • uses a delivery vector to introduce the gene
          • usually modified viruses
            • viral genome is replaced with plasmid
            • virus inserts the gene into the host as part of its normal life cycle
            • problems
              • viruses can indiscriminately insert copies of gene into undesirable regions
                • oncogenesis
              • cells must be dividing for viral integration
                • adenovirus vectors do not require active division, but do not actually insert into the genome
        • can be performed
          • ex vivo
            • cells removed from the body, modified, and transplanted back into the body
          • in vivo
            • cells modified in the body without removal
        • has been used to treat SCID caused by deficiency in the IL-receptor γ chain
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