Lesson 1

• General Outline of Recombinant DNA

  1. Cutting or cleavage of DNA by restriction enzymes (REs)

  2. Selection of an appropriate vector or vehicle which would propagate the recombinant DNA

  3. ligation (join together) of the gene of interest with the vector (cut bacterial plasmid)

  4. Transfer of the recombinant plasmid into a host cell (that would carry out replication to make huge copies of the recombined plasmid)

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

  6. Sequencing of the gene to find out the primary structure of the protein
• Use of molecular techniques to modify the traits of a target organism


  Genetic Engineering
• Some methods to screen Recombinant cells

  1. Selection of plasmid DNA containing cells

  2. Selection of transformed cells with the desired gene

  3. PCR (Polymerase Chain Reaction) detection of plasmid DNA

  4. Genetically Modified Organisms (GMOs)
• Ways in which Plasmids may be introduced into host organisms

  1. Biolistic

  2. Plasmid insertion by Heat Shock Treatment

  3. Electroporation
• Flavr-Savr Tomato was the first genetically modified organism licensed for human consumption where the trait modified in this tomato is its ripening process
• Bt-Corn incorporates the production of a toxin (Bt-endotoxin) from Bacillus thuringiensis in corn plants that results in the death of pests that feed on these plants like corn borer larvae
• A technique that uses a "gene gun" to fire DNA-coated pellets on plant tissues


  Biolistic
• Process that transfer plasmid DNA into bacteria 


  Heat Shock Treatment
•  The modification of traits may involve:

    A. Introduction of new traits into an organism.B. Enhancement of a present trait by increasing the expression of the desired gene.C. Enhancement of a present trait by disrupting the inhibition of the desired genes' expression.
• Cells that survive bombardment, and are able to take up the expression plasmid coated pellets and acquire the ability to express the designed protein.
• In Heat Shock Treatment, The target cells are pre-treated before the procedure to increase the pore sizes of their plasma membrane
•   Afterwards, a "Heat Shock" is done on the plasmid-cell solution by incubating it at 42°C for 1 minute then back to 4°C for 2 minutes.
•  It is believed that the rapid rise and drop of temperature will increase and decreade the pore sizes in the membrane.
•   Electroporation is a method commonly used for insertion of genes into mammalian cells.
•  A selection marker within the inserted plasmid DNA sequence allows the selection of "transformants". Usually an antibiotic resistance gene (e.g. AMP or ampicillin resistance gene) is included in the plasmid DNA. This allows only "transformed" cells to survive in the presence of the antibiotic like ampicillin.
• Blue-white screening protocol


  This is able to screen for cells that were transformed with the desired gene in the cloning site.
• Certain inserted genes within the plasmids provided visible proof of their presence. These include the antibiotic resistance genes that allow for the selection of the transformed cells within the solution.
• PCR reactions specific for the desired gene may be done using DNA from cells.
• It practices focus on the mating of organisms with desirable qualities.

  Classical Breeding
• It confirms the presence of the desired gene in the inserted plasmids.

  PCR Amplification
• A gene for an enzyme that causes the degradation of pectin in the cell walls - meaning normally softens the fruit as it ripens.

  Polygalacturonase
• PCR reactions specific for plasmid sequences will also confirm/ identify the type of plasmid used for the transformation.
• Some of the genetic modifications that promise higher product yield for their targets:

  1 The Flavr-Savr (“Flavor Savor”) tomato

  2 Bt-Corn
• With the ability to insert gene sequences and comes the possibility of providing new traits for these target organisms allows the development of Genetically Modified Organisms or GMOs
• This involves the collection of gene sequences in accessible locations, such as databases (e.g. Genbank (www.ncbi. nlm.nih.gov); Protein Data Bank (www.pdb.org)). 


  Web based research
• Some researchers may be interested in determining if a given gene/trait is available in a particular organism.

  Detection
• It is an in-vitro method that simulates DNA replication in vivo.


  PCR amplification
• PCR Amplification utilizes a thermostable (heat-resistant) DNA polymerase that builds single stranded DNA strands unto unwound DNA templates.
• 
  

  It is by PCR that involves the design of primers that would only bind to sequences that are specific to a target. 


  Gene Detection
• Primers may be classified as

  1 Forward Primers

  2 Reverse Primers
• Ways of PCR Amplification

  1 Detection

  2 Cloning and Expression
• These are complementary and bind to the reverse complementary or (non-coding) sequence of the gene.


  Forward Primers
• These are complementary and bind to the coding sequence of the gene.

  Reverse Primers
• It is may be used to detect the presence of a desired gene in an organism.

  PCR
• Depending on the primer design, the expected product may represent only a specific region of the gene or the entire gene itself.
• This allows the “transformed” bacteria to now produce human insulin as a product. 


  Cloning and Expression
• Certain types of bacteria are capable of cloning and expression since they are able to take genes within their cell membranes for eventual expression.
• Genes that are normally in the form of small and circular DNA structures

  Plasmids
• The genes found in the inserted plasmid DNA sequence will be expressed as proteins that provide specific traits to the transformed bacteria.
• The direct amplification/copying of a full gene is part of the process for "cloning” that gene.