Sequencing

Require:
1. Lots of DNA of interes
2. Way to examine the sequence
  1. Sequencing
    1. sequencing or dideoxy sequencing
      1. Materials
        DNA template
        Taq Polymerase
        dNTPs
        single DNA primer
        If 2: both primers extend at same time, resulting in production of multiple bands: Cannot determine sequence
        ddNTPs (no 3'-OH group)
        If added to polymer during replication, nothing more can be added
        50% chance of adding a dNTP or ddNTP if dNTP and ddNTP are added in equal amounts
        4 tubes:
        1) ddATP, dTTP, dCTP, dGTP
        2) ddTTP, dATP, dCTP, dGTP
        3) ddCTP, dATP, dTTP, dGTP
        4) ddGTP, dATP, dTTP, dCTP
      2. Process:
        1) Denature 2 strands of DNA by heating
        2) Anneal primer
        lower temp
        3) synthesize DNA
        Heat
Polymerase Chain Reaction (PCR)
1. PCR steps
  1. 1) Initiation
    1. 30 sec at 94-96 C
      1. Denature dsDNA to ssDNA
        1 min at 55-65 C
        annealing from cooling - primers bind
  2. 2) Elongation
    1. 1 min at 72 C
      1. synthesis and extension
  3. 3) Termination
    1. 30 sec 94-96 C
    2. Require three cycles to get desired product
      1. Intermediate products
      2. Final Product
2. Materials
  1. DNA you want to amplify: template
  2. Multiple copies of 2 DNA primers
    1. Forward Primer
      1. Extend L to R
    2. Reverse Primer
      1. Extend R to L
    3. 15-20 nucleotides long
  3. dNTPs
  4. DNA polymerase
    1. Taq polymerase
      1. Organisms in heat vents, so can withstand high temp without denaturing
    2. adds nucleotide to 3'-OH group
    3. Reads template 3' to 5'
  5. Thermocycler
3. Agarose Gel Electrophoresis
  1. DNA staining
    1. Ethidium Bromide or SYBR Safe
      1. Flouresce under UV
  2. Agarose: pore size depends on concentr of agarose
  3. Load DNA into wells
    1. Apply electric field
      1. DNA moves from - to +
        DNA is negatively charged
        Shorter pieces of DNA travel further
        Molecular ruler or DNA ladder - mixtures of DNA fragments of known sizes

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Sequencing

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