Genetic manipulation

• Building Blocks of Life

  The study of the DNA
• Genetic Manipulation


  The manipulation of the DNA
• Techniques


  • Cloning
  • Hybridisation
  • PCR
  • Sequencing
  • Gel electrophoresis
• Molecular biology


  Studies macromolecules and the macromolecular mechanisms found in living things
• Molecular biotechnology


  Use of laboratory techniques to study and modify nucleic acids and proteins for applications
• To be able to use the genes for our purpose, we need tools to isolate, identify and characterize them
• Genetic manipulation


  The formation of new combinations of heritable material by the insertion of nucleic acid molecules into a host organism
• Cloning refers to processes used to create exact (genetic) copies of DNA fragments, cells, or organisms
• Basic techniques to manipulate genetic material

  • Purify DNA
  • Manipulate DNA in the lab
  • Produce large amounts of DNA
• DNA is a double helix made of 4 nucleotides
• DNA purification


  1. Precipitation of DNA using alcohol

  2. Freeze & crush

  3. Detergent

  4. Shake with beads
• DNA purification – column with DNA-binding resin


  1. Freeze & crush

  2. Detergent

  3. Shake with beads

  4. Wash away everything not needed

  5. Elute DNA
• Paul Berg’s gene splicing experiment


  1971
• Stanley Cohen introduced a plasmid, pSC 101, into E. coli, which conferred Tetracycline resistance


  1970
• Herbert Boyer discovered that EcoRI enzyme can cut DNA with staggered ends


  1972
• Cohen and Boyer generate the first recombinant organism


  1972
• Restriction endonucleases


  Enzymes that cleave DNA at restriction sites
• Restriction endonucleases were discovered by Werner Arber, Daniel Nathans, and Hamilton Smith in 1978
• Restriction enzymes recognize a palindromic sequence and cleave within it
• DNA ligase


  An enzyme that will repair nicks between adjacent nucleotides
• Recombinant DNA technology - simplified

  1. Cut DNA with restriction enzymes

  2. Insert foreign DNA with plasmid vector

  3. Into host genome

  4. Transgenic organism expresses inserted gene(s)
• Quality control involves monitoring the success of the reaction
• Monitoring cutting/joining reactions


  1. Check the recombinant plasmid cut the products again using the same restriction enzyme

  2. Separate and visualize the fragments produced using gel electrophoresis
• Gel electrophoresis


  A technique that uses a gel made of agarose polymer to separate a mixture of nucleic acids based on size
• Types of cloning vector

  • Plasmid vectors
  • Lambda phage vectors
  • Cosmids
  • Expression vectors
• Cloning vectors


  Self-replicating DNA used to carry the gene of interest into a host system
• A plasmid used to clone a foreign gene is called a cloning vector
• Cloning refers to processes used to create copies of

  • DNA fragments
  • Cells
  • Organisms
• The copied material with the same genetic makeup as the original is called “clone”
• Reproduction of a recombinant plasmid takes place in a host cell
• The host cell copies the cloned DNA using its own replication mechanism to produce multiple copies of a gene
• Cell types used as hosts

  • Bacteria
  • Yeast cells
  • Mammalian cells
• Host cell selection depends on the choice of the vector/plasmid and the aim of the cloning
• Bacteria transformation was shown by Mandel and Higa
• Bacteria transformation


  1. Heat shock in Ca rich solution

  2. Electroporation
• DNA library


  A collection of DNA fragments
• Types of DNA libraries

  • cDNA library
  • Genomic DNA library
• cDNA library


  Used to identify genes encoding proteins in a specific tissue
• Genomic library


  Used to look at gene structure and to compare genomes between organisms
• DNA probes


  A fragment of DNA or RNA used to detect the presence of nucleotide sequences