Bio 99 Midterm 1 Lec: 9

  1. What can you accomplish by cloning?
    • Express proteins in different organisms
    • ex.) Bacterial Multi-Drug Resistance Pump ----> human cells
    • Express modified versions of proteins
    • Purify proteins 
    • Create DNA libraries
  2. Significance of Molecular Biology Techniques
    • Allow us to genetically manipulate multicellular organisms at a single cell level
    • Allow us to visualize processes that are difficult to observe even with the most advanced microscopes
  3. For cloning purposes, what would be the best restriction enzyme to use?

  4. How do we isolate the DNA fragments of interest (red)?
    restriction enzyme digestion (put on plate and choose which one you want)
  5. What is cDNA?
    copied DNA
  6. cDNA Library Construction
    • 1. Blunt ligate the duplex cDNA into vector
    • 2.
    • a. Blunt ligate RE sites to the ends of the DNA
    • b. Clone the cDNA into vector with the same “sticky ends”
  7. DNA libraries: Value of libraries?
    • •As source material to clone a gene
    • •Identification of a mutated gene (functional complementation)
    • •Genome sequencing
  8. Functional Complemenation
    • •Classical/Reverse genetics
    • –Start with wild type organism ----> mutate to obtain organisms with a particular phenotype
    • •Once you have your phenotype, how can we determine which gene was mutated?
  9. Selective temperature
    used for selection against certain cells unable to survive in this selective temperature. For example, in case of temperature-sensitive mutants permissive temperature will be 230C (all of the cells will form colonies), and 360C will be selective temperature that will select against temperature-sensitive mutants(they will be unable to form colonies).
  10. DNA Sequencing
    • •Sanger sequencing
    • – ddNTPs-dideoxy chain termination method
  11. Illumina sequencing- reversible...
    • Terminator Nucleotides
    • Both fluorescent dye and 3’ OH blocking molecule are removed before next nucleotide addition
Card Set
Bio 99 Midterm 1 Lec: 9