Methods of Genetic Engineering and Analysis

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  • Methods of Genetic Engineering and Analysis
    • PCR (Polymerase Chain Reaction)
      • The automated process of the amplification of specific DNA regions as well as analysis
      • 1. Denaturation
        • DNA sample is placed in the cycler and is heated at 94 degrees celsius to form 2 template DNA strands
        • The breakdown of the hydrogen bonds between base pairs to form single-stranded DNA
      • 2. Annealing
        • Temperature decreased to 50-65 degrees celsius so hydrogen bonds can form between bases and primers
          • Primers added in high conc, so they can anneal to exposed bases
        • Primers are necessary to identify sits where synthesis will take place
        • Primers attach to start of DNA strand and then DNA polymerase continues to add nucleotides to rest of chain
        • Follows same principle of semi-conservative replication
        • Primers may be tagged with fluorescent markers to visualise progress of PCR
      • 3. Extension/ Elongation
        • Temperature is increased to 72 degrees celsius so the DNA polymerase builds up newly synthesised polynucleotides
        • Synthesis of polynucleotides are from 5' to 3' direction
      • causes exponential growth in DNA population
    • Gel Electrophoressis
      • Used for the separation of molecules according to their charge/size
      • The DNA molecules run through a gel with a buffer solution once an electrical current is provided
        • The gel is made of either agarose/ polyacrylamide
      • As DNA has the same charge per mass, it is separated according to DNA fragment size
        • Due to the charged phosphate group on sugar-phosphate backbone
      • negatively charged molecules move towards anode
      • Proteins initially denatured and then SDS added so they are separated according to size
      • Smaller size, further distance
      • Higher charge, further distance
      • DNA molecules move through the small pores formed between the hydrogen bonding of the gel molecules
    • Pyro-Sequencing
      • Process of sequencing DNA, to produce a single-stranded DNA strand
        • Complementary to the DNA being sequenced
      • 1. Restriction enzymes used to cut sequencing strands
        • Fragments formed are 300-800 base pairs long
      • 2. Lengths degraded into single stranded DNA (ssDNA)
        • Template DNA that are imobilised
      • 3. Sequencing primer added
        • Incubated with enzymes
          • DNA Polymerase
          • Luciferase
          • Apryrase
          • Luciferin
          • ATP Sulfurase
      • 4. One activated nucleotide is incorporated into complementary DNA strand
    • Replica Plating
      • 1. Pad of sterile cloth pressed on the surface of agar gel on Master Plate
        • sterile cloth may also be filter paper
        • Some colonies will stick to the sterile cloth
      • 2. Cloth transferred on to 2nd petri dish with 2nd marker to see which colonies survive
      • Used to identify recombinant plasmids
      • If foreign DNA disrupted marker gene, then proper gene product will not be made
    • Recombinant Plasmid
      • 1. Plasmid cut through use of restriction enzymes
        • Each restriction enzyme have their own restriction site
        • endonuclease
      • Used to identify recombinant plasmids
      • A Plasmid that's genome is from two different sources
      • 3. DNA fragments form hydrogen bonds with sticky ends
        • Complementary Base pair rules
      • 4. DNA Ligase bonds the DNA together
  • 2. Restriction enzyme causes the production of sticky ends
    • Sticky ends are exposed bases that are 3-4 bases long
    • Recombinant Plasmid
      • 1. Plasmid cut through use of restriction enzymes
        • Each restriction enzyme have their own restriction site
        • endonuclease
      • A Plasmid that's genome is from two different sources
      • 3. DNA fragments form hydrogen bonds with sticky ends
        • Complementary Base pair rules
      • 4. DNA Ligase bonds the DNA together

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