1.2

• Gel Electrophoresis


  A technique for separating charged particles based on variations in their migration rates
• Gel Electrophoresis


  • Applies an electric field through a mixture to move charged molecules in solution
  • Charge, shape, and size of molecules affect their migration in an electric field
  • Common media include paper, polyacrylamide or agarose gels
• Agarose Gel Electrophoresis (AGE)

  • A qualitative and semi-quantitative technique for analyzing isolated DNA
  • Separates DNA fragments, determines sizes of fragments, and analyzes restriction digestion products
  • Resolves 200 bp to 20 kbp DNA fragments
• Agarose Gel Electrophoresis


  1. DNA molecules become entangled with obstacles in the agarose gel matrix

  2. DNA collapses into a globular form as one arm of the "U" slips away from the barrier

  3. Higher agarose concentrations allow better separation of smaller DNAs, while lower concentrations allow resolution of larger DNAs
• Polymerase Chain Reaction (PCR)


  • An in vitro method that simulates DNA replication in vivo
  • Uses a thermostable DNA polymerase to amplify target DNA sequences
• Polymerase Chain Reaction


  1. Denaturation of DNA template at ~95°C

  2. Annealing of primers to ssDNA template at ~54-60°C

  3. Elongation of ssDNA strands by DNA polymerase at ~72°C

  4. Repeated cycles of denaturation, annealing, and elongation to exponentially amplify target DNA sequence
• Polymerase Chain Reaction


  • Thermostable DNA polymerase withstands repeated cycles with little enzyme function loss
  • Each cycle doubles the amount of target DNA sequence
  • Standard PCR uses ~35 cycles, increasing the original target sequence by ~34 billion times
• PCR Detection


  • Primers are designed to bind only to unique sequences of the target gene
  • Allows researchers to determine if a target gene is present in an organism
• Gel Electrophoresis and PCR are used to identify and analyze DNA, RNA, and proteins
• PCR (Polymerase Chain Reaction)


  1. Denaturation of template (95°C)

  2. Primer annealing (54°C)

  3. New DNA strand elongation (72°C)

  4. Repeat steps 1-3 for N cycles
• Polymerase


  • Thermal stability enables it to withstand repeated denaturation, annealing and extension cycles with little enzyme function loss
• The sum of the target sequence is doubled by any cycle of PCR
• A standard PCR experiment uses approximately 35 amplification cycles
• This increases by 235 (i.e. ~34 billion) times the original sum of the target sequence
• Primers


  Designed to only bind to sequences unique to a target
• Primer design


  1. Look at available sequences for the target gene in databases

  2. Align sequences to identify conserved and non-conserved regions

  3. Design forward and reverse primers
• Forward primers


  Complementary and bind to the gene's reverse complementary (non-coding) sequence
• Reverse primers


  Complement and bind to the gene's coding sequence
• PCR Amplification Steps


  1. Undenatured template (54°C)

  2. Template denaturation (95°C)

  3. Primer annealing (54°C)

  4. New DNA strand elongation (72°C)
• Each new dsDNA strand is made up of one old strand from the original template, and one new strand that was generated as a reverse complement of the template
• This is called semiconservative replication of the sequence
• The expected PCR product size depends on the positions at which the forward and reverse primers bind
• PCR Applications


  • Detect presence of a desired gene in an organism
  • Amplify a particular region of a gene
  • Amplify the entire gene for cloning