Week 1

• Biotechnology is the manipulation of living organisms or their components to produce useful products such as pest-resistant crops, new bacterial strains, or novel pharmaceuticals
• Impacts of biotechnology include:

  • Production of medicines
  • Diagnostics
  • Therapeutics like monoclonal antibodies, stem cells, gene therapy, and vaccines
  • Agricultural biotechnology for pollution control, industrial, and marine applications
• Biotechnology revolutionizes medicine by developing life-saving drugs, gene therapies, and personalized medicine, transforming healthcare
• Biotechnology in agriculture provides tools for farmers to make production cheaper and more manageable, such as engineered crops with enhanced traits
• Biotechnology offers sustainable solutions for environmental challenges, including bioremediation and waste management
• Discovery of penicillium:

  • Discovered by Alexander Fleming in 1929
  • Mold with antibacterial agent
  • Useful products obtained from microbial culture have been limited to compounds naturally synthesized by microorganisms
• Genomic DNA:

  • Chromosomal DNA
  • Very long (>20kb fragments)
  • Eukaryotic DNA contains introns
  • Used for genome mapping and sequencing
• Complementary DNA (cDNA) is used to reverse transcribe RNA to DNA:

  • mRNA is purified and used with a Poly-T primer
  • Synthesized from mRNA using Reverse transcriptase enzyme
  • Produces single-stranded cDNA
  • Second-strand synthesis requires primers, nucleotides and DNA polymerase
  • Intron-less (artificial) gene
• Basic steps in gene cloning:

  1. Insert gene-containing DNA fragment into circular DNA molecule (vector) to produce recombinant DNA

  2. Vector transports gene into a host cell, usually a bacterium

  3. Vector multiplies within the host cell, producing numerous copies of the gene

  4. Host cell divides, passing copies of recombinant DNA to progeny, further replication occurs

  5. Clone of identical host cells is produced
• Hosts of biotechnology:

  • Eukaryotic cells: Yeasts, filamentous fungi, mammalian cells, insect cells
  • Prokaryotic cells: Bacteria
• Restriction enzymes cut DNA at specific sites defined by nucleotide sequences:

  • Recognize sequences of 4, 6, or 8 nucleotides
  • Leave overhanging ("sticky") single-stranded tails or blunt ends
  • Useful in the laboratory for cutting DNA at specific sites
• DNA ligase joins DNA fragments in vitro to produce recombinant DNA molecules:

  • Joins 5' phosphate to 3' hydroxyl in DNA
  • Uses ATP
• Plasmid vectors contain:

  • Origin of replication for plasmid replication
  • Selectable marker for antibiotic resistance
  • Cloning site for foreign DNA insertion
• Blue-white screening:

  • Identifies recombinant clones in E. coli
  • Cells with disrupted lacZ gene produce white colonies
  • Cells with normal pUC8 plasmid produce blue colonies
• Cloning supplies large amounts of DNA for gene structure and expression studies
• Gene libraries are collections of clones likely to contain every gene in an organism
• Hybridization probing allows comparison of different cDNAs in colonies:

  • Colonies transferred to a membrane and treated to remove contaminating material
  • DNA is attached to the membrane through sugar-phosphate backbones
  • Labelled probe is applied to the membrane for nucleic acid hybridization
• What primers are required for reverse transcription of RNA to DNA?

  dATP, dGTP, dCTP, dTTP
• What is transformation?

  DNA into prokaryotes (bacteria)
• what is molecular cloning?

  Inserting a DNA fragment of interest
• what are plasmids?

  Small circular DNA molecules that replicate independently in bacteria without being associated chromosomal DNA
• What are the three important features of a plasmid vector?

  Origin of replication

  selectable marker

  cloning, or restriction enzyme, cleavage site
• why is a bacterium DNA not cleaved by restriction enzyme?

  methylation
• describe the uptake of DNA by bacterial cells

  ice-cold salt solution enhances ability to up take DNA efficiently
• How can self-ligation be minimised

  treating vector with phosphatase
• What does lacZ' code for

  enzyme B-galactosidase
• what does x-gal do

  converts substrate x-gal into blue coloured product
• What is Hybridisation probing?

  Allows to compare different cDNA is showed in different colony.
• Process of hybridisation probing

  1. Colonies transferred to a nylon membrane
  2. all contaminating material cells is removed
  3. DNA attached to membrane
  4. labelled with radio active marker
  5. filter is washed
• Agarose gel electrophoresis is used to separate DNA molecules based on size
• Nucleic acids are negatively charged due to their phosphate backbone, causing them to migrate towards the positive electrode
• The gel acts as a sieve, selectively retarding the movement of larger molecules
• Ethidium bromide fluoresces under UV light as it incorporates into DNA
• Visualizing DNA bands under ultraviolet (UV) irradiation depends on the composition of the gel, determining the size of DNA molecules that can be separated
• A 0.5cm thick slab of 0.5% agarose with large pores is used for molecules in the size range of 1 – 30 k
• Techniques to study gene/DNA structure (sequence) include:

  1. Restriction Mapping/Restriction

  2. Fragment Length Polymorphism (RFLP)

  3. Polymerase Chain Reaction

  4. Southern Analysis

  5. DNA Sequencing
• Restriction mapping involves mapping the positions of different restriction sites in a DNA molecule
• To construct a restriction map, a series of restriction enzyme digests must be performed, followed by gel electrophoresis
• Information from single and double digests helps map restriction sites accurately
• Restriction mapping is used to estimate the sizes of DNA molecules through gel electrophoresis