Lecture 19 - Recombinant DNA Technologies

Question 1

What are recombinant DNA technologies?

Answer 1

Joining bits of DNA together (sometimes from different species). These are then inserted into an organism to produce (express) a useful protein

Question 2

What is GFP used for?

Answer 2

Acts as a fluorescent marker

Question 3

What are critical elements for recombinant DNA technologies?

Answer 3

Plasmids

Question 4

What are plasmids?

Answer 4

• Circular pieces of dsDNA
• Replicate independently of host’s cell DNA
• Common in bacteria
• Provide a benefit to host e.g antibiotic resistance.

Question 5

What makes a Eukaryotic cell?

Answer 5

Nucleus

Membrane bound organelles

Often multi cellular organisms

Question 6

What makes a Prokaryotic cell?

Answer 6

No nucleus

No membrane bound organelles

Uni-cellular

smaller

Question 7

What are the key components of recombinant DNA plasmids?

Answer 7

1. Origin of replication
2. Antibiotic resistance gene
3. Promoter
4. Selectable marker
5. Restriction sites

Question 8

Why are the origin of replications needed?

Answer 8

Allows for initiation of replication using host DNA polymerase.

Question 9

Why are Antibiotic resistance genes needed?

Answer 9

Provides survival advantage to cells containing plasmid

Question 10

Why are Promoters needed?

Answer 10

Drives expression of your favourite gene in cells with appropriate transcription factor machinery

Question 11

Why are selectable markers needed?

Answer 11

To select for cells that have successfully taken up the plasmid. e.g the GFP gene.

Question 12

Why are restriction sites needed?

Answer 12

Allows ligation of gene of interest into the cloning vector

Question 13

Promoters are?

Answer 13

Highly specific - need different promoter across species even if for same gene.

Question 14

What are restriction enzymes?

Answer 14

Proteins isolated from bacteria that cut DNA
- cuts dsDNA at specific sequences.

Question 15

How is DNA inserted into a plasma?

Answer 15

Restriction enzymes cut specific regions of DNA,
DNA is inserted in gap

Question 16

What joins the DNA strands together after being cut and DNA inserted?

Answer 16

DNA ligase - via phosphodiester bonds

Question 17

What is the name for a plasmid after a viral gene is added?

Answer 17

Vector

Question 18

What is transformation?

Answer 18

Transfer of vectors into bacteria

Question 19

What are the steps of transformation?

Answer 19

1. Transfer of vector into bacteria
2. Leave for time to grow and divide with recombinant vectors
3. Only cells that take up the plasmid with survive creating a pure sample (due to antibiotic resistance gene)

Question 20

What does it mean by universal genetic code?

Answer 20

All organisms read the same codons as the same amino acids

Question 21

What is the significance of the universal genetic code?

Answer 21

Can transfer genes from bacterial cell into human cell, and vice versa.

Question 22

What do eukaryotic genes have that prokaryotic genes do not?

Answer 22

Exons and introns.

Question 23

What would introns in prokaryotic genes cause?

Answer 23

Would lead to a nonsense polypeptide that did not fold into correct tertiary structure

Question 24

What allows for prokaryotic Pre-mRNA to turn into working mRNA

Answer 24

Removal of introns

Question 25

What is cDNA?

Answer 25

Coding sequence DNA - Eukaryotic DNA with introns removed

Question 26

What is the process of mRNA transcribing back into DNA called?

Answer 26

Reverse transcription

Question 27

What enzyme carries out reverse transcription?

Answer 27

Reverse transcriptase

Question 28

Why is cDNA useful?

Answer 28

No introns allows successful translation to a functional protein in prokaryotic cells.

Without introns in sequence, the overall size of the insert is reduced.