Genetic Engineering

• Genetic Engineering

  1. Isolating the desired gene

  2. Use of restriction endonucleases to cut the required gene from the DNA of an organism

  3. Sticky ends regions with unpaired, exposed bases

  4. Easier to insert the desired gene into the DNA of a different organism
• Isolating the mRNA for desired gene

  1. Use of reverse transcriptase to produce a single strand of complementary DNA

  2. Easier to identify the desired gene as a specific cell will make some very specific types of mRNA
• Vectors

  Bacterial plasmids - small circular molecules of DNA separate from the chromosomal DNA that can replicate independently
• Plasmid getting into a host cell

  Plasmid can combine with the host DNA to form recombinant DNA
• Marker gene

  • Antibiotic resistance marker gene
  • Enable scientists to determine that the bacteria have taken up the plasmid
• Inserting DNA fragment into plasmid

  1. Plasmid is cut open by the same restriction endonuclease

  2. Plasmid has complementary sticky ends to the sticky ends of the DNA fragment

  3. DNA ligase forms phosphodiester bonds between the sugar and phosphate groups on the two strands, joining them together
• 2nd marker gene

  • To show that the plasmid contains the recombinant gene
  • If the DNA fragment is inserted successfully, the marker gene will not function
• Transferring the vector DNA into a cell

  1. Cave the bacterial cells + plasmids in a Ca-rich solution + increase the temperature

  2. Causes the bacterial membrane to become permeable + the plasmids can enter
• Electroporation

  1. A small electrical current is applied to the bacteria

  2. Membranes become very porous + so plasmids move into the cells
• Electrofusion (Produce hybrids)

  1. Tiny electric currents are applied to the membranes of two different cells

  2. Fuses the cell together + nuclear membranes of the two different cells to form a hybrid cell containing DNA from both
• Monoclonal antibody

  Produced by a combination of a cell producing one type of antibody with a tumor cell that divides rapidly in culture