Steps in Recombinant Technology

Question 1

The artificial manipulation, modification, recombination of DNA/other nucleic acid molecules - to modify an organism or population?

Answer 1

Genetic Engineering

Question 2

What discovery emerged possibility for rDNA Tech?

Answer 2

Restriction Enzymes

Question 3

Who discovered restriction enzymes?

Answer 3

(Swiss microbiologist) Werner Arber

Question 4

When were restriction enzymes discovered?

Answer 4

1968

Question 5

Most rDNA Tech involves . . . ?

Answer 5

Insertion of foreign DNA into plasmids of common lab strains of bacteria

Question 6

Small rings of DNA, capable of directing protein synthesis, not part of the bacterium’s chromosome

Answer 6

Plasmids

Question 7

What happens if foreign DNA is inserted?

Answer 7

Able to obtain (almost) limitless copies of the (inserted) gene

Question 8

What is Recombinant DNA Technology?

Answer 8

Joining of DNA molecules from 2 diff. species, inserted into a host organism, to produce new genetic combos

Question 9

What’s Recombinant DNA?

Answer 9

DNA piece created by the combo of (at least) 2 diff. DNA strands

Question 10

What’s the FIRST STEP in Genetic Recombinant Technology?

Answer 10

Isolation of Genetic Material

Question 11

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?

Answer 11

1st step: Isolation of Genetic Material

Question 12

What’s the SECOND STEP in Genetic Recombinant Technology?

Answer 12

Restriction Enzymes Digestion

Question 13

What technique reveals the progress of Restriction Enzymes Digestion?

Answer 13

Agarose Gel Electrophoresis

Question 14

Restriction Enzymes Digestion:

Define Agarose Gel Electrophoresis.

Answer 14

Involves running out of the DNA on agarose gel.

Question 15

Restriction Enzymes Digestion:

How does Agarose Gel Electrophoresis happen?

Answer 15

@ application of current, nega-charged DNA travels to the positive electrode. Separated based on size–allowing separating & cutting out digested DNA fragments.

Question 16

Restriction Enzymes Digestion:

Is vector DNA processed similarly to Agarose Gel Electrophoresis?

Answer 16

Yes.

Question 17

What’s the THIRD STEP in Genetic Recombinant Technology?

Answer 17

Amplification Using PCR

Question 18

Amplification Using PCR:

Define Polymerase Chain Reaction.

Answer 18

Method making multiple copies of a DNA sequence.

Question 19

Amplification Using PCR:

What enzyme is used in PCR?

Answer 19

DNA Polymerase - in vitro

Question 20

Amplification Using PCR:

PCR reactions are run on?

Answer 20

Thermal Cyclers

Question 21

Amplification Using PCR:

Components of Thermal Cyclers used in PCR?

Answer 21

1. Template
2. Primers
3. Enzyme
4. Nucleotides
5. DNA cut fragments amplified using PCR, ligated w/ the cut vector

Question 22

What’s the FOURTH STEP in Genetic Recombinant Technology?

Answer 22

Ligation of DNA Molecules

Question 23

Ligation of DNA Molecules:

What are cut with the same restriction enzyme?

Answer 23

The purified DNA & the vector of interest

Question 24

Ligation of DNA Molecules:

Process of joining 2 together using DNA Ligase?

Answer 24

Ligation

Question 25

Ligation of DNA Molecules:

What’s the result of Ligation?

Answer 25

A hybrid of two DNA molecules

Question 26

Ligation of DNA Molecules:

In genetic terms, the intermixing of diff. DNA strands is?

Answer 26

Recombination

Question 27

Ligation of DNA Molecules:

What’s the hybrid called in Recombination?

Answer 27

Recombinant DNA Molecule

Question 28

What’s the FIFTH STEP in Genetic Recombinant Technology?

Answer 28

Insertion of Recombinant DNA Into Host

Question 29

Which step: rDNA is introduced into a recipient cell (mostly a bacterial cell)?

Answer 29

Insertion of Recombinant DNA Into Host

Question 30

Insertion of Recombinant DNA Into Host:

Process where DNA’s introduced into a recipient cell.

Answer 30

Transformation

Question 31

Insertion of Recombinant DNA Into Host:

Do bacterial cells accept foreign DNA easily? Yes or no? Why?

Answer 31

No. They are treated to be competent in accepting new DNA.

Question 32

Insertion of Recombinant DNA Into Host:

What processes may be used in Transformation?

Answer 32

1. Thermal Shock
2. Ca++ Ion Treatment
3. Recombinant DNA Technology

Question 33

What’s the SIXTH STEP in Genetic Recombinant Technology?

Answer 33

Isolation of Recombinant Cells

Question 34

Which step: This transformation process generates a mixed population of transformed and non-transformed host cells.

Answer 34

Isolation of Recombinant Cells

Question 35

Isolation of Recombinant Cells:

For isolation of rCells from non-rCells, what’s employed?

Answer 35

A marker gene of the plasmid vector

Question 36

What’s the SEVENTH STEP in Genetic Recombinant Technology?

Answer 36

Obtaining or Culturing the Foreign Gene Product

Question 37

Obtaining or Culturing the Foreign Gene Product:

How does the alien DNA get multiplied?

Answer 37

When inserted a piece of alien DNA into a cloning vector, then transferred into a bacterial cell.

Question 38

Obtaining or Culturing the Foreign Gene Product:

What’s the ultimate aim?

Answer 38

To produce a desirable protein expression

Question 39

Obtaining or Culturing the Foreign Gene Product:

The cells harboring cloned genes (of interest) are grown where?

Answer 39

A small scale in the lab

Question 40

Obtaining or Culturing the Foreign Gene Product:

What are these cell cultures used for?

Answer 40

Extracting the desired protein - using various separation techniques.