Producing DNA fragments

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  • Created by: 08rmorris
  • Created on: 26-03-15 10:21
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  • Producing DNA fragments
    • The DNA of two different organisms that has been combined in this way is called recombinant DNA
      • The resulting organism is known as a genetically modified organisms (GMO)
    • The process of making a protein using DNA technology of gene transfer and cloning involves a number of stages
      • 1. Isolation
        • Of the DNA fragments that have the gene for the desired protein
      • 2. Insertion
        • Of the DNA fragment into a vector
      • 3. Transformation
        • That is the transfer of DNA into suitable host cells
      • 4. Identification
        • Of the host cells that have successfully taken up the gene by use of gene markers
      • 5. Growth/Cloning
        • Of the population of host cells
    • Using Reverse Transcriptase
      • The genetic information of retroviruses is in the form of RNA. However, they are able to synthesise DNA from their RNA using Reverse transcriptase.
        • It catalyses the production of DNA from RNA, which is the reverse of the more usual transcription of RNA form DNA .
      • The process of using reverse transcriptase to is late a gene
        • A cell that readily produces the protein is selected
        • These cells have large quantities of the relevant mRNA, which is therefore extracted
        • Reverse transcriptase is then used to make DNA from RNA. This DNA is known as complementary DNA (cDNA) because it is made up of the nucleotides that are complementary to the mRNA
        • To make the other strand of DNA, the enzyme DNA polymerase is used to build up the complementary nucleotides on the cDNA template. This double strand of DNA is the required gene.
    • Using Restriction Endonucleases
      • Some bacteria defend themselves by producing enzymes that cut up the viral DNA.
      • - Cuts a DNA double strand at a specific sequence of bases called a recognition sequence
      • - Cuts DNA in a staggered fashion
        • This leaves an uneven cut in which each strand of the DNA has exposed unpaired bases.
          • Cut from a palindromic sequence
            • The recognition sequence is therefore referred to as a 6bp palindromic sequence
              • This feature is typical of the way restriction endonucleases cut DNA to leave 'sticky ends'

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