Genetic engineering 1

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  • 14 Genetic Engineering 1
    • 1) Size separation in a gel
      • 1) Electrophoresis. DNA loaded into a well within a pre-cast gel (porous matrix that acts like a sieve). DNA migrates through as is negatively charged so moves towards positive electrode (anode)
      • Factors affecting dna migration; 1) dna size - smaller move faster 2)gel concentration- dna moves slower in 1.5% gel compared to 0.7% 3) dna shape compacted dna runs faster. Supercoiled dna > linear > circular 4) Gel type. Agarose gels best for fragments of 100 - 20,000 bps. Polyacrylamide gels are used for 10-700bps
    • 2) Transfer gel to membrane (blotting)
      • 2) Blotting by capillary action. DNA gel placed not an alkaline solution to denature double strand (improves binding). Sheet of nitrocellulosemembrane placed on top of gel.
        • Capillary action - ability of a liquid to flow in narrow space in opposition to gravity
        • Pressure applied. Buffer transfer by capillary action from a region of high to low water potential (filter paper). Exchange interactions bind due to opposite charges. Membrane then backed in a vacuum 80 degrees for 2 hours or exposed to uv radiation
      • Blotting by electric field (electroplating) - relies upon a current and a transfer buffer solution to drive protein or nucleic acids onto membrane
        • Cathode | sponge |  sheets of filter paper soaked in transfer buffer | gel | pvdf/nitrocellulose membrane | 3 sheets filter paper soaked | sponge | anode
      • DNA southern blotting, RNA northern blotting, protein western blotting
      • Advantages; quantifying, isolation, size But requires lots of tissue
    • 3) Detection on membrane by a labelled probe
      • DNA/RNA -Relies on principle of hybridisation (when 2 strands bind). Signal strength proportional to stability of hybridisation.stability depends on degree of match.
      • Protein - Relies on principle of specific antigen-antibody interaction. Modified antibodies linked to a reporter enzyme drives a colorimetric reaction and produces a colour when exposed to substrate
        • Primary antibody used first. Several more bind to enhance signal.
    • In situ hybridization
      • Locates specific genes on chromosomes (chromosome painting) Probes labelled with different fluorescent colours allows simultaneous location of many genes
        • RNA  - Important because all tissues have same dna but transcribes different rna subset. Can detect and quantify expression
          • Method; tissue embedded in block. Block thinly sliced. Apply probe. Wash unbound probes off. Detach bound probes.
            • e.g. detect and locate expression of NMDR genes in brain. Different subunits of the same receptor family expressed in different parts of the brain
        • In situ hybridisation of proteins (relies on antigen-antibody interaction). Immunocytochemistry, immunohistochemistry
      • Advantages; tissue distribution of function and changes. However requires tissue processing. Limited by reagents and resolution


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