DNA Replication

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  • Created by: rosieevie
  • Created on: 05-01-17 13:58

Semi-Conservative Model

Each daughter DNA contains 50% of parental DNA - the rest is copied from the template.

  • (Not conservative - 100% or 0% parental)
  • (Not dispersive - mix of parental DNA)

(http://www.brooklyn.cuny.edu/bc/ahp/BioInfo/graphics/DNArep.01.GIF)

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Meselson and Stahl 1958

  • Tried to determine which DNA model was correct:
  • E.Coli (controlled growth, label, quick growth, purify DNA produced)
  • Grew bacteria in 15N isotope - incorperated into DNA
  • Then transfered to 14N isotope medium
  • DNA isolated for each generation
  • Seperated DNA using density gradient centrifugation
  • 2 seperate bands = semi-conservative model

(http://upload.wikimedia.org/wikipedia/commons/thumb/f/fb/Meselson-stahl_experiment_diagram_en.svg/2000px-Meselson-stahl_experiment_diagram_en.svg.png)

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Prokaryotic DNA Replication

Replication starts at a fixed point and is bi-directional.

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Eukaryotic DNA Replication

  • Multiple replication forks forming at recognition sites
  • Progressing in a bidirectional fashion
  • DNA polymerase addes new bases in 5'---->3' direction

  • Requires a single-stranded template
  • Must extend from exisiting strange e.g. a primer (made of RNA and created by primase)
  • Leading strand - strand synthesised continulously in 5'--->3' direction
  • Lagging strand - made of short fragments in 5'--->3' direction which are joined together (takes longer)
  • Okazaki fragments - short DNA fragments of lagging strand
  • DNA ligase joins together Okazaki graments using ATP to form phosphodiester bonds (NAD in prokaryotes)
  • When nucleotides attach they release pyrophosphate (2 phosphate groups)
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Enzymes in Eukaryotic Replication

  • Helicase - opens up replication fork by breaking hydrogen bonds
  • DNA tropisomerases - distrangles intertwined DNA so it can be acessed
  • ssDNA binding protein - stabalises DNA
  • DNA polymerase III - adds new nucleotides, forming phosphodiester bonds
  • DNA polymerase I - removes RNA primers on lagging strand
  • Ligase - forms phosphodiester bonds between Okazaki fragments
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DNA Sequencing

Sanger Method

  • Uses DNA polymerase to extend primer bound to ssDNA
  • Reactions contain usual dNTPs plus small amount of ddNTP which is missing -OH at 3' carbon
  • ddNTP binding stops reaction as there is no -OH to attatch phosphate to = termination
  • Adding small amount of certain ddNTP e.g. ddATP,ddCTP stops DNA replication at certain points
  • Denature and seperate strands using polyacrylamide gel electrophoresis
  • Reactions with all ddNTPs to create bands that can be read and sequenced

Next Generation Sequencing

  • Short DNA fragments
  • Polymerase chain reaction to amplify copies
  • Reversible fluorescent nucleotides (identification) added
  • Reversible blocking group is used that can be cleaved off using electricity
  • Continuation of PCR and addition of next dNTP
  • Computer algorithms can detect fluorscent signals and contruct a sequence
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