DNA replication

• DNA replication


  1. Produces two identical copies of a DNA molecule

  2. Occurs before cell division
• Helicase


  Unwinds DNA double helix by breaking the hydrogen bonds
• Gyrase


  Prevents supercoiling
• DNA primase


  Inserts an RNA primer onto DNA strands
• DNA polymerase I


  Removes RNA primer, inserts DNA nucleotides
• DNA polymerase III


  Attaches to RNA primer, adds DNA nucleotides (5'-3'), proofreading
• Ligase


  Joins Okazaki fragments
• Semi-conservative DNA replication


  • New DNA molecules have one parent stand and one newly synthesized strand
  • Highly accurate due to complementary base pairing
• PCR (Polymerase Chain Reaction)


  1. Amplifying DNA sequences

  2. Denature - heated to 95 to break hydrogen bonds

  3. Annealing - 54, DNA primers bind to both DNA strands next to the sequence to be copied

  4. Extension - 72, taq DNA polymerase replicates both strands, starts at the primer

  5. Repeat
• Taq DNA polymerase


  • Comes from hot spring bacteria
  • Does not denature in PCR temps, DNA polymerase from humans would
• PCR materials


  • Amplified DNA
  • Buffer
  • Primer - attaches to DNA to be copied
  • Taq DNA polymerase - attaches to prime, creates new DNA strand
  • DNA nucleotides - linked by taq DNA polymerase
• STRs and VNTRs (tandem repeats)

  • Short sequences of DNA
  • Differ slightly between individuals
  • Allows a specific DNA fingerprint to be produced for each person
  • VNTR: 100+ base pairs repeated a few times
  • STR: 2-6 base pairs repeated many times
• Gel electrophoresis


  1. Separates charged molecules

  2. Used to produce a DNA profile/fingerprint

  3. DNA dissociates to become negatively charged in water

  4. Restriction endonuclease enzymes cut DNA into many charged fragments

  5. They move to the positive electrode
• DNA profiling


  1. Amplified with PCR

  2. Cut with restriction endonuclease

  3. Wells or agar, salt solution

  4. Electricity

  5. DNA moves to the positive electrode, small fragments move faster

  6. Dye makes the pattern visible

  7. Forensics + paternity tests
• Prokaryotes


  • One origin of replication with proceeds in both directions
• Eukaryotes


  • Many initiation points
• DNA replication process

  1. Helicase unzips at point of origin

  2. Gyrase releases strain during uncoiling

  3. DNA primase adds an RNA primer to both strands of DNA

  4. DNA polymerase III attaches to the RNA primer and adds DNA nucleotides

  5. Each strand of DNA acts as a template

  6. DNA polymerase III moves towards the replication fork on the leading strand and away in the lagging strand

  7. Okazaki fragments are formed on the lagging strand

  8. DNA polymerase I replaces RNA primer with DNA nucleotides

  9. Ligase joins Okazaki fragments
• Leading and lagging strands

  • DNA polymerase III can only add nucleotides in the 3' end
  • DNA replication is continuous on the leading and discontinuous on the lagging
• Proofreading


  • Errors in base pairing are rare but possible
  • DNA polymerase III proof reads the DNA chain
  • Removes mismatched bases at the 3' end
  • Replaces them with the correct nucleotides