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PCR + applications - Coggle Diagram

- - - - Higher GC content = Higher T\(_m\)
- - - - DNA unwinds
    - - Relieves stress of unwinding downstream of replication fork
    - - Binds + stabilises unpaired strands
    - - Removes primer
    - - Fills gaps between Okazaki fragments
    - - Seals nick between Okazaki fragments
  - - - Okazaki fragments
- - - - Higher temp needed
    - - Dimethyl sulphoxide (DMSO)
      - Glycerol
      - Proprietary mixes
    - - Touch-down PCR
        
        Start from high annealing temp
      - Some DNA polymerases deal with high GC targets better than others
- - - - Template material
        
        Genomic/plasmid DNA, synthetic DNA fragments, whole cells
      - 2 oligonucleotide primers
        
        Forward + reverse
      - DNA polymerase
        
        Thermostable
        
        Taq
        
        Slightly error prone
        
        Pfu
        
        High fidelity 3'-5' exonuclease
      - 4 deoxyribonucleotides
      - Cofactor
        
        Mg\(^{2+}\)
    - - Reaction buffer
        
        pH
        
        [salt]
    - - Drive reaction
  - - - 2.Annealing
        
        Elongation
        
        72\(^o\)C
        
        55\(^o\)C
      - 95\(^o\)C
    - - \(N_{DNAmol}(x)=N_{templmol}x2^x\)
        
        \(N_{templmol}\) is no. template DNA molecules
        
        \(N_{DNAmol}(x)\) is no of DNA molecules after \(x\) PCR cycles
        
        Starting from single molecule
        
        30 cycles
        
        1 x 2\(^{30}\) = 1.1x10\(^9\) DNA molecules produced
    - - 5' end is capped where primer binds
      - 3' end is open
        
        Longer than target strand length
      - After 2nd cycle
        
        Both ends are capped to target strand length
        
        Due to the way the primer binds
        
        After 4 cycles
        
        Target length is >50% dominant
        
        After 11 cycles
        
        Target length is >99%
- - - - \(M_w\) = 650 x bp
      - \(N_{av}\) = 6.02x10\(^{23}\)
- - - - 5-15min
        
        Whole cells - extra long denaturation
    - - Temp depends on primers ~55\(^o\)C
        
        30-60secs
        
        Touch down PCR
        
        Start at 68\(^o\)C + lower by 5\(^o\)C every cycle
      - Extension x25-35
        
        Final extension x1
        
        72\(^o\)C
        
        10 min
        
        72\(^o\)C
        
        Time depends on DNA polymerase + amplicon length
        
        Taq = 30sec/kb
        
        Pfu = 2min/kb
        
        Min 30 sec