Chapter 9 Flashcards
What are some applications for genetic engineering?
- to produce medically/commercially valuable proteins
- to produce specific DNA sequences
- as a tool for studying gene function and regulation
What is genetic engineering?
the deliberate modification of the characteristics of an organism by manipulating its genetic material.
What is a restriction enzyme?
Enzyme that cuts DNA molecule at a specific spot
How is a restriction typically Palindromic?
They cut both strands of DNA at the “same” site so they will have the same sequence only in different directions
What way does DNA move on gel electrophoresis?
Migrates towards (+)
What are a few uses for genetically engineered bacteria?
- treating anemia, dwarfism, diabetus
- making cheese
- making pharmaceuticals
Why would we want to cut the DNA of interest and the plasmid (vector) with the same restriction enzyme?
So they are complimentary
What is a reporter gene?
- gene that researchers attach to a sequence of another gene of interest in order to track it
- Similar to a selective gene
Ex. Green fluorescent protein
What are the functions of a DNA library?
- isolates genomes
- inserts fragments into vectors
DNA genes of interest are introduced to host cell through ________.
Transformation
What is a selectable marker?
A gene that allows cells to grow in otherwise inhibitory conditions
Ex. Cells that take up ampicillin marker can resist the antibiotic and grow on the media
What is a second marker used for?
To distinguish between cells that contain the recombinant plasmid and those that don’t
Ex. Intact vector- grows blue
Recombinant molecule- white
What is the role of DNA ligase in forming a recombinant molecule?
After the “sticky ends” of the gene of interest come in contact with the restriction site on a cell the enzyme covalently closes the plasmid(vector).
What effect does the spliced gene of interest have on the host cell?
Interrupts normal process & the cell no longer functions properly
What happens before the plasmid (vector) & DNA of interest can be introduced into the host cell?
They must be cut with the same restriction enzyme & joined together as a recombinant molecule.
Why is the second marker important for selecting for recombinant molecules?
- not all cells receive the gene of interest.
- some close without taking in any DNA.
- some covalently close around regular DNA
How can you visualize PCR?
Gel electrophoresis
What are the 3 steps to the amplification cycle?
- Heat to 95 to denature DNA
- Lover temp to 50 for primers to adhere
- Reheat to 72 so DNA synthesis can occur
What defines the length of PCR?
Primers
They get shorter each cycle
What is the function of a DNA probe?
To locate specific nucleotide sequences
What is a DNA probe composed of?
Single-stranded piece of DNA
What is the purpose for colony blotting?
To identify which clonies contain a specific sequence of interest
What are the 5 steps in colony blotting?
- Colonies begin on agar plate
- Colonies are transferred to nylon membrane
- Soak membrane in alkaline solution to lyse cells & denature DNA
- Add probe to bind to the DNA of interest
- Probe reveals the colonies with probe of interest
If the new DNA is inserted within the host cell, the second marker is
Inactivated
