Lecture 3 - Recombinant DNA Technology Flashcards
Where does the restriction endonucleases cut at?
- Specific sites in the double-stranded DNA molecule
- Restriction endonuclease cleaves a phosphodiester bond generating two DNA molecules with a 3’-OH and 5’-PO4
What is the function of the DNA ligase?
It’s the enzyme that catalyzes the formation of a phosphodiester bond between nucleotides
How can complimentary ends of two DNA molecules be covalently joined together?
by the enzyme DNA ligase
What does a cloning vector do?
Fools the bacterial host, commonly E.coli, into replicating the foreign DNA fragment
What does the cloning vector and mini-chromosome, plasmid do?
It replicates autonomously in the bacterial host. The presence of the plasmid in the bacterium is detected by the bacterium’s resistance to an antibiotic.
What has 20-40 copies per cell that replicate autonomously, but contains only a few genes?
Plasmid DNA
What are the characteristics of plasmid cloning vectors?
- they occur naturally in various micro-organisms, e.g., bacteria and some yeasts
- some are constructed for recombinant DNA technology, e.g., pBR322
- stably inherited in an extra-chromosomal state, e.g., autonomous replication of the DNA molecule
- frequently carry genes that confer antibiotics resistance on the bacterial host
- posses unique restriction endonuclease recognition sequences employed as cloning (insertion) sites for foreign DNA
- Vary in size from 1-200 Kbp. For cloning, plasmid vectors are small (
What are antibiotics?
drugs that specifically inhibit a metabolic process to impede growth or kill bacterial cells, but not human cells
What is the cloning of DNA in a plasmid?
the construction of a recombinant DNA molecule
What is the selection of cells transformed by recombinant plasmids based on?
Their resistance and sensitivity to specific antibiotics.
