L5-6: Recombinant DNA technology

Question 1

What is recombination of DNA?

Answer 1

When 2 pieces of DNA are brought together under artificial conditions to perform a modified function

Question 2

What are the steps of recombinant DNA technology?

Answer 2

1. Creation of recombinant DNA (new combinations of unrelated genes)
2. Cloning of recombinant DNA (Amplification using PCR)
3. Using recombinant DNA (expressing a cloned gene to produce a protein)

Question 3

What is required to carry form recombinant DNA?

Answer 3

Enzymes, vectors, DNA/RNA and cells

Question 4

What type of cells may be used to make recombinant DNA?

Answer 4

Yeast, bacterial, insect and mammalian cells

Question 5

What enzymes are required to form recombinant DNA?

Answer 5

Restriction enzymes, DNA ligase, Taq polymerase and Reverse transcriptase

Question 6

How are restriction enzymes used?

Answer 6

Cleave DNA at specific sequences, naturally produced by bacteria (used to cleave bacteriophages), many recognise a 4-8bp palindromic sequence

Question 7

What are the 2 types of cleavage that restriction enzymes produce?

Answer 7

Symmetrical cleavage (blunt ends)
Asymmetrical cleavage (sticky ends)

Question 8

How is DNA ligase used?

Answer 8

To anneal the sticky ends together to reform the backbone of DNA

Question 9

What are characteristics of vector DNA that are needed?

Answer 9

Unique restriction sites
Efficient origin of replication
Gene to allow selection of cells that contain the plasmid
Regulatory sequences to allow expression of the inserted gene

Question 10

What are the most commonly used vectors and why?

Answer 10

Plasmids - circular DNA that naturally occur, replicate independently of bacterial chromosome, many different functions, modified for use in genetic engineering

Question 11

What other vectors could be used?

Answer 11

Bacteriophages and Cosmids/ phagemids

Question 12

How is the recombinant DNA transformed into the host?

Answer 12

Use heat shock and CaCl and incubate for 30 minutes

Question 13

What are the advantages of bacteria expression systems?

Answer 13

They are simple, have short generation time, have large yield of products, are low cost

Question 14

What are the disadvantages of bacteria expression systems?

Answer 14

Eukaryotic proteins can fail to fold correctly so lose biological activity, proteins can be toxic to bacteria, no post-translational modification

Question 15

What are advantages of yeast expression systems?

Answer 15

They are simple unicellular eukaryotes, resemble mammalian cells, grow quick and cheap, perform post-translational modifications

Question 16

What are disadvantages of yeast expression systems?

Answer 16

Contains proteases which can degrade some recombinant proteins, post-translational modifications may differ in mammalian cells

Question 17

What are advantages of insect cell expression systems?

Answer 17

High-level protein expression, correct folding of mammalian proteins, post-translational modifications and cheaper than mammalian cell culture

Question 18

What are disadvantages of insect cell expression systems?

Answer 18

Post-translational modifications may differ from mammalian cells

Question 19

What are advantages of mammalian expression systems?

Answer 19

Best place to produce mammalian proteins, correct folding of mammalian proteins, post-translational modifications

Question 20

What are advantages of mammalian expression systems?

Answer 20

Complex cells, grow to lower cell densities and are expensive

Question 21

Why was it disadvantageous to extract insulin from animals?

Answer 21

Not identical to human, difficult to purify and possible contamination with pathogens