Lesson 2:Evolution

• Genetic Engineering (GE) is the set of technologies used to alter the genetic makeup of cells. It includes transfer of genes within and across the species to produce novel organisms.
• Genetic Engineering has numerous applications in research, agriculture, industrial biotechnology and medicine.
• Recombinant DNA


  A DNA molecule produced by combining DNA from different sources
• Steps in obtaining recombinant DNA

  Cutting-Ligation-Insertion-Isolation

  
• Restriction enzyme


  DNA-cutting enzyme that recognizes one or a few target sequences and cuts DNA at or near those sequences

  
• EcoRI


  A common restriction enzyme that cuts at the sequence G/AATTC, producing 5 nucleotide sticky ends with 5' end overhangs of AATTC

  
• Plasmid

  A small, circular, double-stranded DNA molecule that is distinct from a cell's chromosomal DNA and naturally exists in bacterial cells

  
• General steps of recombinant DNA technology

  1. Isolating DNA

  2. Cutting or cleavage of DNA by restriction enzymes

  3. Selection of an appropriate vector

  4. Ligation of the gene of interest with the vector

  5. Amplifying the recombinant DNA

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

  7. Sequencing of the gene to find out the primary structure of the protein

  
• Biolistics

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


  A process used to transfer plasmid DNA into bacteria by increasing the pore sizes of their plasma membranes
• Electroporation


  A technique similar to Heat Shock Treatment but the expansion of the membrane pores is done through an electric "shock"
• Methods to screen recombinant cells

  1. Selection of plasmid DNA containing cells

  2. Selection of transformed cells with the desired gene
• Cells transformed with plasmids containing a disrupted β-galactosidase gene will not turn blue in the presence of IPTG

  
• Transformation of cells


  Allow plasmid-containing cells to grow and propagate into colonies
• Selection of transformed cells with the desired gene

  1. Antibiotic resistance genes allow for selection of transformed cells

  2. Inserted genes produce colored or fluorescent products that label the colonies/cells
• Blue-white screening


  1. Insertion of gene in cloning site disrupts β-galactosidase gene, preventing colored product formation

  2. Cells transformed with disrupted gene remain "white" in presence of IPTG

  
• PCR detection of plasmid DNA

  1. PCR amplification confirms presence of desired gene

  2. PCR reactions identify type of plasmid used for transformation

  
• Genetically Modified Organisms (GMOs)

  • Ability to insert gene sequences provides new traits
  • Some GMOs promise higher product yield (e.g. Flavr-Savr Tomato, Bt-Corn)

  
• Concerns have been raised regarding consumption of GMO foods
• Manufacturers are urged to provide labels that notify consumers of GMO presence in their products
• GMOs are believed to be safe when licensed by food regulatory agencies
• Consumers must be provided with enough information to make their own choices regarding use of GMOs
• Genetic Engineering


  The deliberate modification of the characteristics of an organism by manipulating its genetic material
• Central Dogma


  An explanation of the flow of genetic information within a biological system developed by James Watson and Francis Crick to describe the process of protein production: DNA to RNA to protein
• DNA (Deoxyribonucleic acid)


  An organic acid and polymer composed of four nitrogenous bases - adenine, thymine, cytosine, and guanine, intervening units of phosphate and the pentose sugar deoxyribose
• Gene


  A portion of DNA that contains instructions for making a protein
• Genome


  The complete set of genetic information in an organism. It provides all the information the organism requires to function. In living organisms, the genome is stored in long molecules of DNA called chromosomes
• Gene Mapping


  The sequential allocation of loci to a relative position on a chromosome
• Genetic maps


  Species-specific and comprised of genomic markers and/or genes and the genetic distance between each marker
• Recombinant DNA


  Molecules of DNA from two different species that are inserted into a host organism to produce new genetic combinations that are of value to science, medicine, agriculture, and industry
• Biotechnology


  Technology based on biology - biotechnology harnesses cellular and biomolecular processes to develop technologies and products that help improve our lives and the health of our planet
• Plasmids


  A genetic structure in a cell that can replicate independently of the chromosomes, typically a small circular DNA strand in the cytoplasm of a bacterium or protozoan. Plasmids are much used in the laboratory manipulation of genes
• Polymerase Chain Reaction (PCR)


  A laboratory technique used to make multiple copies of a segment of DNA
• Cloning


  The process of generating a genetically identical copy of a cell or an organism
• Gene Therapy


  The insertion of genes into an individual's cells and tissues to treat a disease, and hereditary diseases in which a defective mutant allele is replaced with a functional one
• Vector


  A DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed (e.g., plasmid, cosmid, Lambda phages)
• Host Cell


  A cell that has been introduced with DNA (or RNA), such as a bacterial cell acting as a host cell for the DNA isolated from a bacteriophage