Amplifying DNA fragments

• Amplifying fragments of DNA

  Making lots of copies of the isolated fragments of DNA
• Amplifying DNA fragments


  1. In vivo

  2. In vitro
• In vitro amplification


  1. Fragments of DNA are amplified via the polymerase chain reaction (PCR)

  2. This is conducted by an automated machine
• PCR


  Polymerase chain reaction
• Equipment list to carry out in vitro

  • Thermocycler
  • DNA fragment to be amplified
  • DNA polymerase - Taq polymerase
  • Primers
  • DNA nucleotides
• Primers


  Short pieces of DNA which are complementary to the bases at the start of the DNA fragment
• How PCR can be used to amplify DNA fragments

  1. A reaction mixture which contains the DNA sample, free nucleotides, primers and DNA polymerase

  2. DNA mixture is increased to 95 degrees to break hydrogen bonds and split the DNA into two strands - denaturing

  3. The temperature is then decreased/ cooled to 55 degrees so the primers can attach - annealing

  4. The reaction temperature is increased to 72 degrees - the optimum temperature for taq DNA polymerase

  5. The enzyme DNA polymerase attaches complementary free nucleotides and makes a new strand to attach to each template

  6. Two new copies of the fragment of DNA are produced and one cycle of PCR is complete

  7. The cycle begins again - mixture heated to 95 degrees and now all four stands (two new and two old) are used as templates
• Advantages of PCR


  • Automated and therefore efficient
  • Rapid - had the ability to make 100 billion copies of DNA within hours
  • Doesn't require any living cells so it's quicker and less complex
• In vivo amplification


  Fragments of DNA are amplified inside a living organism
• Promoter region


  • Added to the start of the DNA fragment
  • This is a sequence of DNA that is a binding site for RNA polymerase
  • Enables transcription
• Terminator region


  • Added to the end of the DNA fragment
  • Causes RNA polymerase to detach
  • Stops transcription of the fragment
• Vectors


  DNA molecules used to carry DNA fragments into host cells
• Examples of commonly used vectors

  • Plasmids (small, circular molecules of DNA in bacteria)
  • Bacteriophages (Viruses which infect bacteria)
• Use of restriction endonucleases in inserting fragments of DNA into vectors


  1. Used to cut open the vector DNA

  2. The same restriction endonuclease to create the DNA fragment is used on the vector DNA
• Why the same restriction endonuclease enzyme is used in the fragment and the vector


  To ensure the sticky ends on the vector is complementary to the DNA fragment
• Use of DNA ligase in inserting fragments of DNA into vectors


  Used to stick together the sticky ends of the DNA fragment to the DNA vector
• Ligation


  DNA ligase ligates the fragments and the vector
• Recombinant DNA


  Vector DNA + DNA fragment
• Transformation of host cells using these vectors

  1. Mixing the bacteria cell (host cell) in a solution of calcium cells

  2. These sudden changes to the temperature make the bacterial membrane more permeable and the plasmid (vector) enters
• Potential issues with transformation

  • The recombinant plasmid/ vector doesn't get inside the cell
  • The plasmid re-joins before the DNA fragment entered
  • The DNA fragment sticks to itself, rather than inserting into the plasmid
• Identification of genetically modified (GM) cells or organisms

  1. To identify of the plasmid was taken up - the bacteria (host cell) grown on agar plates containing antibiotics - ampicillin

  2. Identifying if the plasmid had taken up the recombinant plasmid - the DNA fragment is inserted in the middle of marker gene
• Why the DNA fragment is inserted in the middle of a marker gene


  That protein will no longer be made so the bacteria won't be resistant to another bacteria or it will not produce fluorescent protein
• How identification of recombinant DNA occurs


  1. The surviving cells in the ampicillin can be tested further

  2. Those that do fluoresce under UV light must contain a non-recombinant plasmid

  3. Those that do not fluoresce must have the recombinant plasmid

  4. The bacteria containing recombinant DNA have been identified

  5. These bacteria can be grown and the DNA fragment is therefore amplified
• Examples of different types of marker genes

  • Antibiotic resistance genes
  • Genes coding for fluorescent proteins
  • Genes coding for enzymes
• Growth of host cell

  • Once identified colonies of bacteria which contain DNA fragments can be produced in Large quantities
  • This is done through the use of a fermenter
  • Large cloned population of host cells can produce the protein coded for by the inserted DNA fragment - through the universal mechanisms of transcription and translation