GenBio2SumTest1

• Genetic engineering


  a gene modification process wherein the Deoxyribonucleic acid (DNA) is transferred from one organism to another.
• Classical breeding


  Focuses on the mating of organisms with desirable qualities or traits
• Genetic engineering


  Involves molecular techniques to modify the traits of a target organism.
• Genetically modified organisms


  Have been subject for public scrutiny whether it is safe to use or ethically accepted.
• 4 Processes of Genetic Engineering

  DNA Cleavage, Production of Recombinant DNA, Cloning, Screening
• Stage 1. DNA Cleavage


  A restriction endonuclease is used to cleave the source DNA into a different set of fragments. The endonuclease’s recognition sequence is likely to occur many times within the source of DNA, thus cleavage will produce a large number of different fragments. The fragments can be separated from one another according to their size by gel electrophoresis.
• Stage 2. Production of Recombinant DNA


  The fragments of DNA are inserted into plasmids or viral vectors that have been cleaved with the same restriction endonuclease as the source DNA.
• Stage 3. Cloning


  The plasmids or viruses serve as vectors that can introduce the DNA fragments into cells--- usually, but not always bacteria. As each cell produces, it forms a clone of cells that all contain the fragment-bearing vector.
• Stage 4. Screening


  The clones containing a specific DNA fragment of interest are identified from the clone library
• Methods of Introducing Plasmids into the Host Organism

  Biolistics, Plasmid insertion by Heat Shock Treatment, Electroporation
• Methods to Screen Recombinant Cells

  Selection of plasmid DNA containing cells, Selection of transformed cells with the desired gene, Polymerase Chain Reaction (PCR) detection of plasmid DNA
• PCR reactions specific for plasmid sequences will confirm/identify the type of plasmid used for the transformation through the following steps:

  Step 1. Denaturation, Step 2. Annealing of Primers, Step 3. Elongation
• Biolistics


  This technique uses a “gene gun” to fire DNA-coated pellets on plant tissues. Cells that are able to survive and take up the expression plasmid coated pellets can acquire the ability to express the designed protein.
• Plasmid insertion by Heat Shock Treatment


  is a process used to transfer plasmid DNA into bacteria
• Electroporation


  This technique follows a similar methodology as Heat Shock Treatment but uses electric shock to expand the membrane pores. This method is commonly used for the insertion of genes into mammalian cells.
• Selection of plasmid DNA containing cells


  A selection marker within the inserted plasmid DNA sequence allows the selection of “transformants”.
• Selection of transformed cells with the desired gene


  The most general procedure for screening clone libraries to find a particular gene is hybridization - the cloned genes form base-pairs with complementary sequences on another nucleic acid.
• Polymerase Chain Reaction (PCR) detection of plasmid DNA


  the presence of the desired gene in the inserted plasmids may also be confirmed through PCR amplification
• Step 1. Denaturation


  An excess primer, synthetic sequence of 20 to 30 nucleotides
• Step 2. Annealing of Primers


  The solution is allowed to cool to about 60°C so that the single strands of DNA reassociate into double strands.
• Step 3. Elongation


  Using the primer, the polymerase copies the rest of the fragment and both DNA strands are replicated resulting into two copies of the original fragment