Bio Q3 - Genetic Engineering

• Breeding - controlled process of subjecting two parent organisms to sexual reproduction to produce offspring.
• Classical Breeding - natural process
• Genetic Engineering - involves the direct manipulation of the genes of an organism to express the desired trait: also known as Recombinant DNA Technology.
• Examples of genetic engineering/recombinant DNA technology

  • Bt Corn (Bt toxin gene from Bacillus thuringiensis to combat corn borers.
  • Transgeniczebrafish or GloFish inserted with green fluorescent protein (gfp) gene
• Importance of genetic engineering

  • Pest-resistant crops - potatoes can be GM'd to combat pests like Colocado beetle
  • Frost-resistant crops - GM'd lettuce to resist to adverse environmental condition
  • Plants with extra vitamins - Golden Rice for more beta-carotene
• Selective Breeding - a process wherein people select and breed only those that have desirable traits; also known as artificial selection
• Recombinant DNA - molecules of DNA from two different species inserted into a host organism.
• DNA - Deoxyribonucleic acid
• Components of rDNA (recombinant DNA):

  1. Vector Gene - vehicle; used to carry foreign DNA into another cell
  2. Donor Gene - inserted into a vector gene to be replicated
• Plasmid - circular, double-stranded, extra chromosomal DNA molecules
• Characteristics of Plasmid:

  • self-replicating; replicons
  • easy to modify
  • useful as a vector
• Parts of a plasmid

  • Replicating Zone - DNA sequences recognized by replication enzymes of the host cell
  • Screening Zone - genes that have antibiotic resistance; R factor
  • Cloning Zone (Polylinkers) - segments of DNA that contain multiple sites for restriction enzyme digestion
• Transgenic Organism -organism created through genetic engineering, contains DNA from other source
• Restriction Enzyme/Endonuclease - cuts DNA at specific recognition sites
• Palindromic Sequence - DNA sequence that read the same forward and backward; recognition sites that provides substrate for restriction endonucleases
• History of Genetic Engineering:

  • (1972) - Stanley Cohen and Herbert Boyer introduced a plasmid segment into Escherichia coli (first transgenic organism).
  • Werner Arber pioneered the study of restriction enzymes
  • Hamilton Smith and Daniel Nathans explained restriction enzymes.
• Process of GE

  1. Isolation
  2. Cutting of DNA
  3. Ligation
  4. Transformation/Cloning
  5. Screening
• Isolation - isolation of both plasmid and donor gene
• Cutting of DNA - cleaved by restriction enzymes to have sticky ends
• Sticky ends - DNA fragments with overlapping sides which have unpaired nucleotides at its ends
• Ligation - plasmid and donor gene are mixed with the use of DNA ligase which creates a phosphodiester bond.
• DNA ligase - enzymes that rejoin DNA fragments
• Transformation/Cloning - plasmid is inserted to the host and then cultured via culture media
• Ampicillin is mixed to eliminate bacterial cells that didn't take up recombinant plasmid
• Screening - identification of desired cloned gene

  • Colony Hybridization - method of identifying a bacterial colony that contains the gene of interest.
  • Oligonucleotide Probe - detest presence of nucleotide sequences that are complementary to the sequence in the probe.
  • Autoradiography - technique using X-ray film to visualize molecules or fragments of molecules that have been radioactively labeled.
• Application and Issues on rDNA Technology

  1. Agriculture - crops, meat, fish, etc.
  2. Bioremediation - use of microbes to degrade environmental contaminants
  3. Pharmaceuticals - making medicine; insulin, alprolix, human growth hormone, factor IX, factor VII, interferon
  4. Genetic Testing - detection of genetic disorders, especially at birth; detection of Huntington’s disease
  5. Gene Therapy - treatment of genetic disorders in humans; viruses are used as vectors