Ch 9: Biotechnology and Recombinant DNA Flashcards
What is the function of restriction enzymes?
- Cut DNA at defined positions close to or within their recognition sequence
- Typically recognize 4, 6, or 8 base sequences
True or false. Blunt ends of DNA can only ligate with specific blunt ends.
False. Any blunt ends can ligate together
True or false. Stick-end DNA overhangs must be complementary in order to ligate.
True
Why do bacteria produce restriction enzymes?
They restrict the ability of foreign DNA (such as bacteriophage DNA) to infect/invade the host cell by cleaving it
How is host DNA modified to protect it from its own restriction enzymes?
Methylation of their sequences at C or A nucleotides
What E. coli plasmid vector is commonly used for cloning?
pUC19
What protein does the gene lacZ encode for?
β-galactosidease
What protein does the gene ampR encode for?
Ampicillin resistance
What stain is used to visualize DNA on an agarose gel?
Ethidium Bromide
What are some methods of transformating DNA into cells in the lab? (5)
- Electroporation
- Chemical transformation
- Protoplast fusion
- Gene gun
- Microinjection
What is the difference between transformation and transfection?
Transformation: uptake of DNA into a prokaryotic cell; term means something different in mammalian cells
Transfection: uptake of DNA into mammalian cells
True or false. Most cell types don’t naturally transform.
True
How does electroporation work?
- Controlled short, but powerful electrical pulse induces temporary hydrophilic pores in in the cell membrane
- DNA can enter the cell
- Applicable to all cells
- Organisms with cell walls may require conversion to protoplast
Explain how chemical transformation works in E. coli
- Cells incubated in ice-cold calcium chloride
- DNA is added
- Mild heat shock
Explain how chemical transformation works for yeast
- Incubated in ice-cold lithium chloride
- DNA added
- Mild heat shock
Rates of protoplast fusion increase in the presence of _____
Polyethyleneglycol (PEG)
How does a gene gun (or biolistic transformation) work?
- Microscopic gold or tungsten particles (1 μm) coated with DNA propelled into cells with burst of helium
- If a particle lands on the nucleus the DNA may be incorporated into the chromosome
In what organisms is biolistic transformation typically used? (3)
- Plant cells
- C. elegans
- Yeast mitochondria