Steps in Recombinant Technology Flashcards
The artificial manipulation, modification, recombination of DNA/other nucleic acid molecules - to modify an organism or population?
Genetic Engineering
What discovery emerged possibility for rDNA Tech?
Restriction Enzymes
Who discovered restriction enzymes?
(Swiss microbiologist) Werner Arber
When were restriction enzymes discovered?
1968
Most rDNA Tech involves . . . ?
Insertion of foreign DNA into plasmids of common lab strains of bacteria
Small rings of DNA, capable of directing protein synthesis, not part of the bacterium’s chromosome
Plasmids
What happens if foreign DNA is inserted?
Able to obtain (almost) limitless copies of the (inserted) gene
What is Recombinant DNA Technology?
Joining of DNA molecules from 2 diff. species, inserted into a host organism, to produce new genetic combos
What’s Recombinant DNA?
DNA piece created by the combo of (at least) 2 diff. DNA strands
What’s the FIRST STEP in Genetic Recombinant Technology?
Isolation of Genetic Material
DNA w/ other macromolecules are within a cell membrane together, they need to be separated and purified and involves enzymes in order to. Which step?
1st step: Isolation of Genetic Material
What’s the SECOND STEP in Genetic Recombinant Technology?
Restriction Enzymes Digestion
What technique reveals the progress of Restriction Enzymes Digestion?
Agarose Gel Electrophoresis
Restriction Enzymes Digestion:
Define Agarose Gel Electrophoresis.
Involves running out of the DNA on agarose gel.
Restriction Enzymes Digestion:
How does Agarose Gel Electrophoresis happen?
@ application of current, nega-charged DNA travels to the positive electrode. Separated based on size–allowing separating & cutting out digested DNA fragments.
Restriction Enzymes Digestion:
Is vector DNA processed similarly to Agarose Gel Electrophoresis?
Yes.
What’s the THIRD STEP in Genetic Recombinant Technology?
Amplification Using PCR
Amplification Using PCR:
Define Polymerase Chain Reaction.
Method making multiple copies of a DNA sequence.
Amplification Using PCR:
What enzyme is used in PCR?
DNA Polymerase - in vitro
Amplification Using PCR:
PCR reactions are run on?
Thermal Cyclers
Amplification Using PCR:
Components of Thermal Cyclers used in PCR?
- Template
- Primers
- Enzyme
- Nucleotides
- DNA cut fragments amplified using PCR, ligated w/ the cut vector
What’s the FOURTH STEP in Genetic Recombinant Technology?
Ligation of DNA Molecules
Ligation of DNA Molecules:
What are cut with the same restriction enzyme?
The purified DNA & the vector of interest
Ligation of DNA Molecules:
Process of joining 2 together using DNA Ligase?
Ligation
Ligation of DNA Molecules:
What’s the result of Ligation?
A hybrid of two DNA molecules
Ligation of DNA Molecules:
In genetic terms, the intermixing of diff. DNA strands is?
Recombination
Ligation of DNA Molecules:
What’s the hybrid called in Recombination?
Recombinant DNA Molecule
What’s the FIFTH STEP in Genetic Recombinant Technology?
Insertion of Recombinant DNA Into Host
Which step: rDNA is introduced into a recipient cell (mostly a bacterial cell)?
Insertion of Recombinant DNA Into Host
Insertion of Recombinant DNA Into Host:
Process where DNA’s introduced into a recipient cell.
Transformation
Insertion of Recombinant DNA Into Host:
Do bacterial cells accept foreign DNA easily? Yes or no? Why?
No. They are treated to be competent in accepting new DNA.
Insertion of Recombinant DNA Into Host:
What processes may be used in Transformation?
- Thermal Shock
- Ca++ Ion Treatment
- Recombinant DNA Technology
What’s the SIXTH STEP in Genetic Recombinant Technology?
Isolation of Recombinant Cells
Which step: This transformation process generates a mixed population of transformed and non-transformed host cells.
Isolation of Recombinant Cells
Isolation of Recombinant Cells:
For isolation of rCells from non-rCells, what’s employed?
A marker gene of the plasmid vector
What’s the SEVENTH STEP in Genetic Recombinant Technology?
Obtaining or Culturing the Foreign Gene Product
Obtaining or Culturing the Foreign Gene Product:
How does the alien DNA get multiplied?
When inserted a piece of alien DNA into a cloning vector, then transferred into a bacterial cell.
Obtaining or Culturing the Foreign Gene Product:
What’s the ultimate aim?
To produce a desirable protein expression
Obtaining or Culturing the Foreign Gene Product:
The cells harboring cloned genes (of interest) are grown where?
A small scale in the lab
Obtaining or Culturing the Foreign Gene Product:
What are these cell cultures used for?
Extracting the desired protein - using various separation techniques.
