Genetic Engineering

• Genetic Engineering


  The use of molecular techniques to modify the traits of a target organism
• Modification of traits in genetic engineering

  • Introduction of new traits
  • Enhancement of a present trait by increasing the expression of the desired gene
  • Enhancement of a present trait by disrupting the inhibition of the desired genes' expression
• General outline of recombinant DNA

  1. Cutting or cleavage of DNA by restriction enzymes

  2. Selection of an appropriate vector

  3. Ligation of the gene of interest with the vector

  4. Transfer of the recombinant plasmid into a host cell

  5. Selection process to screen which cells contain the gene of interest

  6. Sequencing of the gene
• Biolistics


  A technique using a "gene gun" to fire DNA-coated pellets on plant tissues
• Heat Shock Treatment


  A process used to transfer plasmid DNA into bacteria by making cells "competent"
• Heat Shock Treatment steps

  1. Pre-treat cells with CaCl2

  2. Incubate with plasmid at 4°C

  3. Heat Shock at 42°C for 1 minute

  4. Return to 4°C for 2 minutes
• Electroporation


  A technique that uses an electric shock to expand membrane pores for gene insertion
• Methods to screen recombinant cells

  • Selection of plasmid DNA containing cells
  • Selection of transformed cells with the desired gene
  • PCR detection of plasmid DNA
• Selection marker


  A gene within the inserted plasmid DNA sequence that allows the selection of "transformants"
• Transformants


  Cells that have taken up plasmid DNA and acquired new traits
• Antibiotic resistance genes are often included in plasmid DNA for selection purposes
• Flavr-Savr Tomato


  The first genetically modified organism licensed for human consumption
• Bt-Corn


  Corn plants developed to produce a toxin from Bacillus thuringiensis to kill pests
• The toxin produced by Bt-Corn is selective for Lepidoptera larvae and is non-toxic to humans, mammals, fish, and birds
• GMOs can provide new traits for target organisms, promising higher product yield
• Concerns have been raised regarding the consumption of genetically modified foods
• Insulin Production


  Insertion of Human Insulin Gene into Bacteria
• Pest Resistance

  Insertion of Bt-toxin gene into Corn/Maize
• Delayed Ripening


  Disruption of a gene for a ripening enzyme in Tomato plants
• Chymosin Production


  Insertion of a gene for chymosin into Bacteria
• PCR amplification


  1. Detection of specific genes in target organisms

  2. Simulates DNA replication in vitro

  3. Uses thermostable DNA polymerase

  4. Involves repeated cycles of incubation at different temperatures
• A typical PCR experiment uses about 35 cycles of amplification
• Each cycle of PCR doubles the amount of the target sequence
• PCR can increase the original amount of the target sequence by 2^35 times
• Gene detection by PCR

  Involves the design of primers that bind to specific sequences of a target
• DNA polymerase


  Enzyme that builds single stranded DNA strands onto unwound DNA templates
• PCR (Polymerase Chain Reaction)


  1. Unwinding DNA template

  2. Annealing of a primer

  3. Extension of ssDNA strand
• PCR uses repeated cycles of incubation at different temperatures
• This increases the original amount of the target sequence by 2^35 (i.e. ~34 billion) times
• Primers


  Short oligonucleotide sequences designed to bind to specific target sequences
• Gene detection by PCR involves the design of primers that would only bind to sequences that are specific to a target
• Designing primers

  1. Look at available sequences

  2. Align sequences for similarity

  3. Design primers for conserved areas

  4. Design primers for non-conserved areas
• Steps in PCR Amplification

  1. Step 0: Undenatured Template

  2. Step 1: Template denaturation

  3. Step 2: Primer Annealing

  4. Step 3: New DNA strand elongation

  5. Step 4: Repeat steps 1 to 3
• Template denaturation


  DNA strands are separated; H-bonds between complementary sequences are broken
• Primer Annealing


  1. bonds are formed between complementary sequences on the primers and the target sequences
• New DNA strand elongation

  Two new dsDNA strands are formed by elongation of generated ssDNA
• Semiconservative replication


  Each new dsDNA strand is made up of one old strand and one new strand
• The expected product of PCR amplification will depend on the sequences/position at which the primer sequences bind
• Successful insertion of a gene allows the expression of its protein product
• Expression plasmid components

  • Promoter
  • Multiple Cloning Site
  • PCR Primers
  • Inserted Gene Sequence
  • Antibiotic Resistance Gene