Recombinant DNA technology

Question 1

What is the definition of 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 an example of a gene used in recombinant DNA technologies?

Answer 2

Green fluorescent protein (GFP) (from jellyfish)

Question 3

Describe the molecular structure and features of a plasmid (3)

Answer 3

• Derived from endogenous DNA
• Circular, double stranded
• Replicate independently from host’s chromosomal

Question 4

What are 4 key components of a plasmid?

Answer 4

• Origin of replication (ORI)
• Antibiotic resistance gene
• Promoter
• Restriction site

Question 5

What is the role of the origin of replication?

Answer 5

allows initiation of replication using host DNA polymerase

Question 6

What is the role of the antibiotic resistance gene?

Answer 6

allows selection of the cells which have take up the protein/plasmid (all others killed by antibiotic)

Question 7

What is the role of the promotor?

Answer 7

Drives expression of your favourite gene (e.g. insulin or GFP) in cells with appropriate transcription factor machinery

Question 8

What is the role of the restriction site?

Answer 8

so that you can put what you want into the backbone

Question 9

What are 2 tools used for recombinant DNA technologies?

Answer 9

1. Cutting and pasting DNA into plasmids
2. Amplifying plasmids - transformation

Question 10

What 2 things are used for cutting and pasting DNA into plasmids?

Answer 10

restriction enzymes and DNA ligase

Question 11

What is the general function of restriction enzymes and where are they naturally found?

Answer 11

○ Naturally found in bacteria
○ Used to stop invading pathogens/phages entering the cells
= defence system to degrade foreign DNA

Question 12

What do restriction enzymes do in terms of recombinant DNA technologies?

Answer 12

Cuts dsDNA at specific sequences
- Has to be double stranded and unmethylated (= from hosts)
- Cuts so that there’s overhang = sticky ends

Question 13

What are sticky ends used for?

Answer 13

Add to what we want to insert to plasmid with corresponding sticky ends, then repair DNA

Question 14

What does DNA ligase do in terms of recombinant DNA technologies?

Answer 14

Catalyses the formation of phosphodiester bond to repair nick in DNA backbone

Question 15

What is transformation?

Answer 15

transfer of plasmids into bacteria

Question 16

Which bacteria do we transform?

Answer 16

the bacteria selected by antibiotic resistance contained on plasmid

Question 17

What is the difference between using a plasmid with a bacterial gene vs with a eukaryotic gene when inserted into bacteria?

Answer 17

The gene will be expressed if bacterial, only replicated lots if eukaryotic

Question 18

What is the difference between prokaryotic and eukaryotic genes, and what does this mean for prokaryotes?

Answer 18

Prokaryotic genes don’t have introns, and thus prokaryotes don’t have the machinery to process eukaryotic introns

Question 19

What is the significance of having a universal genetic code?

Answer 19

• All organisms read the same codons as the same amino acids
.: we can transform a human gene into bacteria and it will still make the same protein

Question 20

To use prokaryotes to replicate a eukaryotic gene, which part(s) of the gene are used?

Answer 20

coding sequence only!

Question 21

When and why is it best to transform the plasmid into bacteria?

Answer 21

• Bacteria are best if don’t need post-translational modification/folding
• Fast and cost-effective

Question 22

If the protein of interest requires post-translational modification, what are the best cells to use?

Answer 22

Mammalian, transgenic

Question 23

what are examples of post-translational modification? (2)

Answer 23

cleaving C chain, glycosylation

Question 24

What cells are used to make EPO? Why?

Answer 24

CHO cells - very good at glycosylating proteins

Question 25

What protein is goat milk used to produced? What is its function?

Answer 25

anti-thrombin (AT) - Blocks the function of thrombin, which is in the clotting cascade

Question 26

How is AT produced?

Answer 26

milk specific promotor added to human AT gene (is a mammary cell specific protein so AT not produced in every cell for whole life). AT is then produced in the milk and purified out