GER lecture 2

• In vitro

  "In a glass" or "in a test tube" - You're in control of all conditions of the reaction
• In vivo

  "In the living" - Reaction conditions are the same as the organism's natural conditions
• Ex vivo

  "Outside the living" - External environment but with minimal alteration of natural conditions
• Gel-based assays separate and analyse molecules (DNA, RNA and proteins) and their fragments based on their size and charge (more charge moves faster through gel)
• Agarose gel electrophoresis

  Network of pores through where DNA molecules travel towards the +ve pole
• Agarose gel electrophoresis

  • Low % agarose has large pores, best for separation of large DNA fragments
  • High % agarose has small pores, best for separation of small DNA fragments
  • Smaller molecules travel faster and further through the gel
  • Supercoiled DNA runs a lot faster than linear DNA
  • Nicked DNA is "relaxed" and bulky (doesn't run fast)
  • DNA dye is needed to visualise DNA using a UV light
• SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)

  Polyacrylamide polymerises in contact with water and forms a network of pores through which the molecules travel towards the +ve pole
• SDS-PAGE

  • Low % acrylamide has large pores, best for separation of large DNA fragments
  • High % acrylamide has small pores, best for separation of small DNA fragments
  • Smaller molecules travel faster and further through the gel
  • SDS is a detergent added to remove higher order structures and separate the protein fragments based on length only
  • Gel is stained to visualise proteins
• Denaturing conditions


  Used to analyse the primary structure of molecules - Natural structure is disrupted and the protein unfolds into a linear chain and bound proteins dissociate
• Native conditions


  Used to analyse the natural structure of molecules - Non-denaturing conditions preserve the molecule's natural structure
• Electro mobility-shift assay (EMSA)


  Solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel
• EMSA experiments

  • EMSA-CAP
  • EMSA-Taq polymerase
• EMSA-CAP

  CAP binds to DNA and leads to high levels of transcription, but CAP can only bind to DNA in the presence of cAMP
• Denaturing polyacrylamide gel

  Sequencing gel that resolves DNA in very high resolution (separates base pairs)
• Footprinting


  Technique to find where proteins bind to DNA by using DNA-cleaving enzymes (DNase and exonuclease) - Wherever the DNA is not cleaved is where the protein is bound
• Footprinting with DNase


  1. DNase cuts at various locations within the DNA

  2. Bound protein protects DNA from cleavage

  3. There is a section of the gel that is missing, which is where the DNA was not cleaved because of the protein
• Footprinting with exonuclease


  1. Exonuclease can only cleave from one end of DNA in a 3' → 5' direction

  2. Need to do the experiment twice, once from each direction, and label the opposite strand
• Examples of denaturing agents include:

  • Urea for DNA
  • SDS for proteins