lesson 3

• Recombinant DNA technology involves modifying and recombining DNAs to produce desired products like proteins, animals and plants with desirable traits
• Genetic engineering


  Process of altering the DNA in an organism's genome and hence alter the organism's characteristics (phenotype) in a particular way
• Genetic engineering may involve changing one base pair, deleting a whole region of DNA, introducing an additional copy of a gene, or extracting DNA from another organism's genome and combining it with the DNA of that individual
• Genome


  An organism's complete set of DNA
• Phenotype


  An individual's observable traits, such as height, eye color, and blood type
• Genotype


  Refers to the genetic material passed between generations
• Recombinant DNA Technology


  Technique used in genetic engineering that involves the identification, isolation and insertion of "desired" gene (target DNA) into a vector such as a plasmid or bacteriophage to form a recombinant DNA molecule and production of large quantities of that gene fragment or product encoded by that gene
• Recombinant DNA technology has wide-ranging applications such as medicine, forensics, pharmaceuticals, and agriculture (improving livestock and crop production)
• Tools used in Recombinant DNA Technology

  • Target DNA: gene of interest
  • Restriction Enzymes: cut DNA into fragments
  • DNA Cloning Vectors: carry target gene into host cell
  • Host Cell: bacterial cell that allows cloning vector to replicate within it
  • Modifying Enzymes: DNA Ligase and Taq polymerase
• Stages in Gene Cloning

  1. Isolation of a target gene

  2. Insertion of a target gene into a vector

  3. Introduction of a vector into a host

  4. Amplification of the target gene by the host cell (cloning) and screening
• Restriction Enzymes


  Naturally found in certain bacteria that resistant to bacteriophage infections, function as a defense mechanism that protects the bacteria by cutting the bacteriophage DNA at specific base sequences
• Plasmids


  Small, often circular DNA molecule found in bacteria and other cells that exist independently of the host chromosome and are generally non-lethal
• Bacteriophages


  Viruses that affect bacteria, exist independently of bacterial chromosomes but rely on the bacterial machinery for replication
• The target DNA extracted and cleaved enzymatically by restriction endonuclease enzymes are then ligated (joined) by the enzyme ligase to vector DNA to form a recombinant DNA molecule
• Suitable host cells like E. coli, as well as yeast and fungi are selected and the recombinant DNA molecule is introduced into these host cells through the process of Transformation
• Those cells that have successfully taken up the recombinant DNA molecules are called transformed cells (or recombinant cells), and are separated from the untransformed cells by using selection methods
• Stages in Gene Cloning


  1. Introduction of vector into a suitable host (Transformation)

  2. Selection of transformed host cells
• Stages in Gene Cloning


  Amplification of the target gene by the host cell (cloning) and screening
• Recombinant DNA technology involves the joining (mediated) by the enzyme ligase to vector DNA to form a rDNA molecule
• Stages in Gene Cloning

  • Stage III: Introduction of vector into a suitable host (Transformation)
  • Stage IV: Amplification of the target gene by the host cell (cloning) and screening
• Transformation


  The process of inserting rDNA into the host cell
• Transformed cells (or recombinant cells)


  Cells that have successfully taken up the rDNA molecules
• The transformed cells are separated from the untransformed cells by using various methods that use marker genes
• It must be ensured that the foreign DNA inserted into the vector DNA expresses the desired character in the host cells
• The transformed host cells must also be able to be multiplied to obtain sufficient number of copies
• If needed, such genes may also be transferred and expressed into another organism
• Polymerase Chain Reaction (PCR)


  A technique used in molecular biology to make many copies of (amplify) small sections of DNA or a gene
• PCR


  • Enables the generation of thousands to millions of copies of a particular section of DNA from a very small amount of DNA
  • Common tool used in medical and biological research
• Core "ingredients" of PCR

  • DNA template to be copied
  • Primers
  • dNTPs (DNA nucleotide bases)
  • Taq polymerase enzyme
  • Buffer
• Polymerase Chain Reaction (PCR)

  1. Denaturing

  2. Annealing

  3. Extending
• Genetic engineering can be used to produce plants that have a higher nutritional value or is able to resist parasitic attacks or can tolerate exposure to herbicides
• Genetic engineering has been applied to improve the resilience, nutritional value and growth rate of crops, e.g. rice, tomatoes, potatoes
• Transgenics


  One or more DNA sequences from another species have been introduced by artificial means
• Ethical issues in genetic engineering

  • Health risks and long-term effects on environment of transgenics and genetically modified foods
  • Whether certain research should be banned or morally impermissible
  • Whether chimeras and transgenics are more likely to suffer than "traditional" organisms
  • Whether these interventions will redefine what it means to be "normal"
  • Whether chimeric entities possessing degrees of intelligence should be given rights and special protection
  • What social and legal controls should be placed on such research
  • What unintended personal, social or cultural consequences could result
  • Who will have access to these technologies and how will scarce resources be allocated