DNA/Protein Analysis and Sequencing

Separating and Analyzing DNA

  • Agarose Gel Electrophoresis
    • Similar to SDS-PAGE but uses agarose, a different polymer better suited for separating large DNA macromolecules.
    • Small DNA fragments run faster and lower on the gel due to less obstruction.
    • DNA samples are loaded into wells, and an electric charge is applied to make the DNA move.
    • DNA, possessing a negatively charged phosphate backbone, migrates towards the positive charge.
  • Visualization
    • Ethidium bromide (or safer alternatives like cyber safe) intercalates into the DNA.
    • This changes the UV absorption and emission properties allowing visualization under UV light.

Sanger Sequencing Basics

  • Uses dideoxynucleotides (ddNTPs) which lack a 3' hydroxyl group, causing chain termination upon incorporation.
    • ddNTPs are tagged with different fluorescent colors for each base (A, G, C, T).
    • Incorporation of a ddNTP halts DNA synthesis at that point, creating fragments of varying lengths.
    • Fragments are separated by size using gel electrophoresis, and a fluorescent reader identifies the terminal nucleotide of each fragment.
    • Generates an electropherogram showing the sequence with corresponding peak intensities.
  • Limitations
    • Typically yields reliable reads of up to 500-800 nucleotides.
    • Cost is approximately $4 per sequence.
    • Commercial services are available, including on-campus facilities like Barker Hall.

Next-Generation Sequencing

  • Capable of whole-genome sequencing and high-depth reads (approximately 400 bases).

Other Methods

  • Northern and Southern Blots: Similar to Western blots, but used for RNA and DNA respectively, utilizing oligonucleotide probes.
  • DNA Footprinting: Identifies DNA sequences bound by a protein.
    • The protein protects the DNA it binds from nuclease digestion.
    • The protected DNA fragment is then sequenced using Sanger sequencing to determine the binding sequence.

Protein Purification Methods Review

  • Purifying Proteins Based on Charge: Uses anion and exchange chromatography with resins of positive or negative charges.
  • Electric Gels: SDS-PAGE and agarose gel electrophoresis
  • Gel Filtration: Separates proteins by size using porous resin; smaller proteins take longer to elute.
    *Analytical: Usually used for analytical purposes to determine if your protein is running like a monomer or a dimer, trimer, tetramer, etcetera.
  • Dialysis: Used to change buffer conditions by allowing salts to pass through a semi-permeable membrane while retaining the protein.
  • Affinity Chromatography: Exploits specific binding properties of proteins, such as using a His-tag and nickel column.
    • A protease can be used to cleave the His-tag after purification.