BGM1004/L05 Recombinant DNA Technology I

Question 1

Give the 3 principles of recombinant DNA technology.

Answer 1

Creation of recombinant DNA
Cloning of recombinant DNA
Using recombinant DNA

Question 2

Describe creation of recombinant DNA. (2)

Answer 2

Construction of new combinations of unrelated genes
2 pieces ligated to perform modified function

Question 3

Describe cloning of recombinant DNA.

Answer 3

Amplifying new DNA many times to introduce into living cells

Question 4

Describe using recombinant DNA.

Answer 4

Expressing a cloned gene to produce a protein

Question 5

Give an overview of how to create recombinant DNA. (3)

Answer 5

Take 2 pieces of DNA from unrelated sources
Use biological environment to clone and amplify
Express cloned DNA to make a protein

Question 6

Give 4 types of host cell.

Answer 6

Yeast
Bacteria
Mammalian cells
Human cells

Question 7

Give an advantage of using yeast and bacterial cells as host cells.

Answer 7

Easy to manipulate

Question 8

Give an advantage of yeast cells over bacterial cells as a host.

Answer 8

Yeast cells eukaryotic so closer to mammalian cells

Question 9

Give a disadvantage to human and mammalian cells as a host.

Answer 9

Difficult to manipulate as they easily become infected

Question 10

Name the 4 enzymes involved in recombinant DNA synthesis.

Answer 10

Restriction enzymes
DNA ligase
Taq polymerase
Reverse transcriptase

Question 11

What do restriction enzymes do?

Answer 11

Cleave DNA at specific sequences

Question 12

What does DNA ligase do?

Answer 12

Ligates fragments of DNA together through phosphate backbone

Question 13

What does Taq polymerase do?

Answer 13

Copies DNA many times

Question 14

What does reverse transcriptase do?

Answer 14

Copies RNA into DNA

Question 15

Give 2 types of cleavage.

Answer 15

Symmetrical
Asymmetrical

Question 16

What enzyme can ligate 2 pieces of DNA cut by the same restriction enzyme?

Answer 16

DNA ligase

Question 17

What 2 types of DNA are required to create recombinant DNA?

Answer 17

Vector DNA
Insert DNA

Question 18

What 5 features must recombinant DNA have?

Answer 18

Unique restriction site
Efficient oriC
Selective gene
Regulatory sequences
Easily introduced into host

Question 19

How long are plasmids?

Answer 19

2-200kbp

Question 20

What is a polylinker?

Answer 20

Insert of DNA with recognition sites for many restriction enzymes

Question 21

Give 2 other examples of vectors.

Answer 21

Bacteriophages
Cosmids
Phagemids

Question 22

Give 2 uses of recombinant DNA.

Answer 22

Expression
Recovery and further manipulation

Question 23

What process is useful for amplifying a specific piece of DNA?

Answer 23

Polymerase Chain Reaction (PCR)

Question 24

How are cells made competent? (2)

Answer 24

Heat shocking and adding CaCl

Question 25

What temperature are cells incubated at for recombinant DNA transformation?

Answer 25

37C

Question 26

How long are cells incubated for recombinant DNA transformation?

Answer 26

30 mins

Question 27

How can transformed cells be selectively cultured?

Answer 27

Grown on a selective medium e.g., containing antibiotic