3.8.4.1 Recombinant DNA technology

• Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another.
• Key stages in producing a transgenic organism:

  • Isolation of required gene in DNA fragments
  • Insertion of gene (DNA fragment) into a vector
  • Transforming host cells
  • Identification of transformed cells
  • Growth of isolated transgenic cells
• Recombinant DNA technology is possible as genetic code is universal, as are transcription and translation mechanisms.

  • transferred DNA can be translated within cells of recipient (transgenic) organism
• Three ways of isolating a gene include using reverse transcriptase, using restriction endonucleases and using a gene machine.
• Fragments of DNA can be amplified by in vitro and in vivo techniques

  • amplifying DNA involves cloning genes so you have many copies
• Polymerase chain reaction (PCR) is an in vitro method to amplify DNA fragments.
• The culture of transformed host cells is an in vivo method to amplify DNA fragments, where transformed cells are grown and divide by mitosis.
• Restriction Enzymes (Restriction Endonucleases) can be used to isolate a desired gene:

  • These enzymes cut DNA at specific recognition sequences (palindromic sequences).
  • They produce sticky ends.
  • This means the ends will not be complementary, so will not join together.
• Reverse Transcriptase can be used to isolate a desired gene:

  • mRNA from a cell that expresses the desired gene is used.
  • Reverse transcriptase converts mRNA (single-stranded) into double-stranded complementary DNA (cDNA).
  • This is useful because introns are removed, making it easier to express the gene in prokaryotes.
• A Gene Machine can be used to isolate desired genes:

  • Artificially synthesises DNA using a computer.
  • Can design sequences free of introns to work in prokaryotic cells.
• Once the gene is isolated, it must be inserted into a vector to transfer it into a new organism.

  Common Vectors:

  • Plasmids
  • Viruses
• How is the Gene Inserted into a Vector?

  • The same restriction enzyme used to cut the gene is used to cut the plasmid, creating complementary sticky ends.
  • The fragments are incubated with the plasmids, if a plasmid takes up the insert, base pairing takes place between the complementary ends
  • DNA ligase joins the sticky ends of the gene and plasmid via phosphodiester bonds, forming recombinant DNA.
• Once the recombinant DNA is ready, transgenic organisms can be formed using electroporation. This is a high-voltage pulse that makes the bacterial membrane more permeable, stimulating bacterial cells to take up plasmids.
• Not all host cells successfully take up the recombinant DNA, so scientists use marker genes to identify transformed cells, to separate them from non-transformed cells. Common Marker Genes:

  • Antibiotic Resistance Genes – Bacteria with the plasmid survive on antibiotic plates.
  • Fluorescent Genes (e.g., GFP from jellyfish) – Cells glow under UV light if transformation is successful.
  • Enzyme Markers – A specific substrate reaction confirms transformation.
• Medical Applications of Recombinant DNA technology:

  • Producing Human Insulin – The insulin gene is inserted into bacteria to mass-produce insulin for diabetics.
  • Gene Therapy – Used to treat genetic disorders.
  • Vaccines – Recombinant DNA allows the production of viral proteins for vaccines.
• Recombinant DNA technology has useful applications in agriculture. It can produce genetically modified crops that are pest-resistant, herbicide-resistant etc. and can produce genetically modified animals that can grow faster for example.
• Benefits of Recombinant DNA technology:

  • Can cure genetic diseases.
  • Increases food production.
  • Reduces the need for pesticides & antibiotics
• Concerns of Recombinant DNA technology:

  • Environmental Risks – GM crops may crossbreed with wild plants.
  • Health Concerns – Some fear GM foods could cause allergies.
  • Ethical Issues – Is it right to genetically modify animals?