Who led the research team that first purified DNA polymerase and developed an in vitro assay for DNA synthesis?
Arthur Kornberg
What is the role of RNA primase in DNA replication?
To lay down RNA primers on the lagging strand template at the replication fork.
What are the two key protein domains of DNA polymerase, and what are their functions?
DNA synthesis domain: Adds nucleotides to the growing strand.
Error-correcting domain (exonuclease): Removes incorrectly inserted nucleotides.
What is the function of DNA ligase in DNA replication?
To join Okazaki fragments on the lagging strand by sealing the phosphodiester backbone between a 3’-OH and a 5’-phosphate of adjacent nucleosides.
What energy molecule does DNA ligase use to seal DNA ends?
ATP
Who is credited with inventing the concept of the Polymerase Chain Reaction (PCR)?
Kary Mullis
What was the key “lightbulb moment” for Kary Mullis in inventing PCR?
Using two opposing primers and repeated cycles of denaturation, annealing, and DNA synthesis to amplify a specific DNA sequence exponentially (2ⁿ).
What was the major practical problem with the original proof-of-concept PCR experiments?
The DNA polymerase was denatured by the high heat (95°C) each cycle, so fresh enzyme had to be added manually after every cycle.
What discovery made PCR scalable, less error-prone, and suitable for automation?
The discovery and use of a heat-stable DNA polymerase.
From what organism was the first heat-stable DNA polymerase isolated, and where was this organism found?
Thermus aquaticus (Taq), isolated from a hot spring in Yellowstone National Park.
Which company filed the key patents for the PCR process and recombinant Taq polymerase, and which company later purchased these rights for $300 million?
Cetus Corporation filed the patents; Roche purchased them.
What are the two main ethical/legal issues raised by the patenting of Taq polymerase? (Name the concepts)
Bioprospecting/Biopiracy: Commercializing a natural resource without fair compensation to the community/country of origin.
Inventorship and priority narratives: Disputes over whether the patent acknowledged prior research and made false claims.
What was the outcome of the 2000 legal challenge to the Taq polymerase patent (‘818 patent’)?
A US federal judge ruled the patent invalid, agreeing with the challenge that the scientists had misrepresented their experiments and falsely claimed advances over previous discoveries (specifically the work of Chien, Edgar & Trela).
