Updated: 11/18/2016

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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