Gene Therapy Overview

Summary of Gene Therapy

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  • DNA Technology
    • In Vivo Cloning
      • Sticky ends will join to other complementary sticky ends
      • DNA ligase used to join phosphate sugar backbone
      • Fragment and vector cut with same restriction endonuclease
        • Sticky ends allow binding
      • Transformation (plasmid taken up by vector)
        • Mixed in a medium containing calcium ions; bacteria becomes permeable
      • Gene Markers
        • Antibiotic Resistance
          • Inserted gene takes the place of one resistance and keeps another
        • Fluorescent Markers
          • Cells that have taken up the gene will glow
        • Enzyme Markers
          • Lactase gene, turns substrate blue
    • In Vitro Cloning
      • Polymerase Chain Reaction
        • 1) 95 degrees celcius; strands separate
        • 2) 55 degrees celcius; DNA primers added- prevents stands rejoining
        • 3) 72 degrees celcius; DNA polymerase join nucleotides - two new strands formed
        • Copies DNA fragments, rapid and efficient
    • Recombinant DNA
      • To increase yield from animals or crop
      • To improve nutrient content of food
      • To introduce resistance
      • To make crop plants tolerant to herbicides
      • To make vaccines
    • Locating and Sequencing Genes
      • DNA Probes
        • DNA probe made of bases complementary to DNA sequence
      • DNA Sequencing
        • 1) Four test tubes set up, each with one terminator nucleotide base, primer,mixture of nucleotides and DNA fragments to be sequenced
        • 2) Random binding of nucleotides will eventually cause fragments to terminate after every base, creating different size fragments
      • Gel Electrophresis
        • 1) Fragments placed in wells in agar gel
        • 2) Voltage applied
        • 3) Resistance of gel means smaller molecules move faster
        • As smaller fragments move further, when sequencing read from bottom to top
      • Restriction Mapping
        • Cutting DNA with a series of different restriction enzymes
        • Fragments produced separated by gel electrophresis
        • This can now be done automatically by a computer
    • Medical Diagnosis
      • Can be used to screen for specific genes
      • Can be used to identify carriers of genes
    • Producing DNA Fragments
      • Using Reverse Transcriptase
        • Using Retroviruses- able to synthesise DNA from RNA using reverse transcriptase
        • 1) isolate
        • 2) Relevant mRNA extracted
        • 3) Reverse transcriptase makes DNA from mRNA - DNA known as cDNA
        • Known as cDNA as it is made up of nucleotides complementary to mRNA
        • To make the other stand DNA polymerase builds up complementary nucleotides
      • Using Restriction Endonucleases
        • Cuts DNA at specific recognition sites
        • Some leave sticky ends - staggered edge; exposed unpaired bases


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