Recombinant DNA technologies

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Created by
Pierre Gasly

Cards (28)

  • Describe how Complementary DNA is synthesised

    • mRNA is hybridized with a Poly-T primer
    • Reverse transcriptase uses dNTPs to produce a single cDNA strand
    • mRNA is degraded using RNAse H
    • DNA polymerase uses primers and dNTPs to produce the second cDNA strand
    • Incubate with Terminal transferase
  • The basic steps in gene cloning
    1. Recombinant DNA produced
    2. Vector transports the recombinant DNA into the host
    3. The vector replicates
    4. The host divides

    The result is a colony containing the recombinant DNA.
  • Restriction endonucleases
    Cut the DNA at specific sites by recognising the nucleotide sequence they leave a free 5' phosphate and 3' hydroxyl group. They may produce sticky or blunt ends.
  • How are DNA fragments joined together
    DNA ligase joins the phosphodiester backbone by ligasing the free 5' phosphate and 3' hydroxyl groups.
  • What is the requirement for a host in genetic technology
    They can be grown easily in large quantities.
    They must be non-pathogenic.
  • What is transformation
    The uptake of DNA by bacteria
  • How is Antibiotic resistance used in genetic technology
    Used to select for bacterial cells that have taken up a plasmid during transformation
  • What colour is a recombinant in Blue-white screening
    White.
  • Gene libraries
    This is a collection of clones that is likely to contain every single gene in a particular organism. They are created by inserting a number of different DNA fragments in the vector.
  • Hybridisation probing
    The colonies are transferred to a nitrocellulose or nylon membrane.
    They are treated so that the DNA is denatured.
    DNA is exposed to UV irradiation and binds to the membrane using the sugar-phosphate backbone.
    The probe is labelled with a radioactive marker which hybridises to the DNA.
    The unbound probe is washed away and the hybridised probe is detected.
  • What are the features of cDNA
    It is described as artificial because it has no introns
  • How is recombinant DNA generated
    A DNA fragment is inserted into a plasmid Vector
  • Why are plasmids used as vectors
    They are small circular DNA molecules which replicate independantly.
  • Label the plasmid and describe its functions.
    The selectable marker is often a gene that codes for antibiotic resistance. This allows fo you to identify which of your clones have taken up the interested gene.
    The restriction enzyme site is a specific DNA sequence that the restriction endonuclease recognises. And so this is where the foreign DNA is inserted.
    A) Origin of replication
    B) Antibiotic resistance
    C) Selected marker
    D) Inserted gene
    E) Promotor
  • How are two fragments cut with the same restiction endocuclase joined together
    Using DNA ligase and ATP
  • How are these two strands joined together as they were cut with different endonucleases
    The sticky ends are filled in with DNA Polymerase using dNTPs.
    Then DNA ligase can join the blunt ends using ATP.
  • What is a competent host
    A host which has undergone an ice-cold Calcium Chloride treatment to allow the plasmid to bind to its exterior.
  • How is transformation increased
    Soaking in an ice-cold salt.
    Heating to 42 C.
  • What are the three possible products of plasmid ligation
    Self ligation.
    Desired recombinant DNA.
    Wrong recombinant DNA.
  • What causes self-ligation of plasmids
    A lack of DNA
  • How is DNA inserted into a plasmid
    The plasmid and DNA are cut with the same endonuclease and then incubated together.
  • What causes the formation of the wrong recombinant plasmid
    The presence of contaminant DNA.
  • How are recombinants identified
    Using blue-white screening.
  • How can self ligation be prevented
    By treating the ligated plasmid with phosphatase.
  • How does phosphatase prevent re-circulation of plasmids
    Removes the 5' Phosphate.
  • How does blue-white screening work
    The colonies are incubated on a plate containing Xgal.
    The recombinant plasmids lacZ gene has been inactivated due to the insertion of the DNA within the gene.
    They are unable to produce Beta galactosidase to convert Xgal into the blue product.
  • What type of sequences do restriction enzymes cut
    Palindromic
  • What is a palindromic DNA
    The sequence of nucleotides on one strand in the 5 to 3 direction is the same as that on the other sequence in the 5 to 3 direction.