Lecture 19: RECOMBINANT DNA TECHNOLOGIES

Question 1

How many people get type 1 diabetes?

Answer 1

1 in 5000

Question 2

When does type 1 diabetes develop?

Answer 2

Childhood usually

Question 3

What is type 1 diabetes?

Answer 3

Loss of beta cells in the pancreas which make insulin

Question 4

How many people get type 2 diabetes?

Answer 4

15%

Question 5

When does type 2 diabetes develop?

Answer 5

Lifelong risk

Question 6

What is type 2 diabetes?

Answer 6

Resistance to insulin

Question 7

What happens in 1922?

Answer 7

Insulin first isolated from pig and cattle pancreas

Question 8

What happened in the 1950’s?

Answer 8

Insulin proteins sequence/structure identified

Question 9

What happened in 1973?

Answer 9

Recombinant DNA technology introduced (PCR and restriction enzymes)

Question 10

What happened in 1977?

Answer 10

Insulin gene mapped to chromosome 11

Question 11

What happened in 1980?

Answer 11

Gene for insulin sequenced

Question 12

What happened in 1982?

Answer 12

Eli Lilly produced first recombinant insulin

Question 13

Where was insulin purified from?

Answer 13

The pancreas of cattle or pigs

Question 14

How does of insulin compare between humans, cows and pigs?

Answer 14

The amino acid sequences are similar but not identical

Question 15

What is the structure of insulin?

Answer 15

Alpha and beta chains held together by disulfide bonds (1 between the two chains and one within the alpha chain)

Question 16

How many differences between human and cow insulin?

Answer 16

2

Question 17

How many differences between human and pig insulin?

Answer 17

1

Question 18

What happens when cow or pig insulin is introduced into humans?

Answer 18

Immune responses range from local irritation to anaphylactic shock

Question 19

What is the problem with cow and pig insulin?

Answer 19

It isn’t always 100% pure

Question 20

What has also been used as therapeutic proteins?

Answer 20

Human sources but there are issues arounds safety (pathogen transmission), yields and source of a protein (tissue availability)

Question 21

What are recombinant DNA technologies?

Answer 21

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

Question 22

What is the structure of plasmids?

Answer 22

Usually circular pieces of double stranded DNA found independently of the chromosome

Question 23

How do plasmids replicate?

Answer 23

Independently of the hosts chromosomal DNA but uses all the cells machinery

Question 24

Where are plasmids found?

Answer 24

Common in bacteria, but also found in eukaryotes and some fungi

Question 25

What do plasmids provide?

Answer 25

A benefit to hosts - antibiotic resistance

Question 26

What are the key components of recombinant DNA using plasmids?

Answer 26

Origin of replication (ORI), antibiotic resistance gene and promoter

Question 27

What does the ORI allow?

Answer 27

Initiation of replication using host DNA polymerase

Question 28

What does the antibiotic resistance gene allow?

Answer 28

Selection of cells containing plasmid

Question 29

What does the promoter do?

Answer 29

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

Question 30

What must the promoter do?

Answer 30

It needs to change to allow expression in prokaryotes, eukaryotes and specific cell types

Question 31

What is used to cut DNA?

Answer 31

Restriction enzymes

Question 32

Where are restriction enzymes found?

Answer 32

Naturally in bacteria, used as a defence system to degrade foreign DNA

Question 33

What do restriction enzymes do?

Answer 33

Cut the double stranded DNA of the plasmid at specific sequences

Question 34

How is the DNA insert made?

Answer 34

To match the cut of the DNA backbone with complementary base pairing

Question 35

What does DNA Ligase do?

Answer 35

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

Question 36

What is transformation?

Answer 36

Transfer of plasmids into bacteria

Question 37

What are transformed bacteria selected by?

Answer 37

Antibiotic resistance contained on plasmid

Question 38

What happens after the transformed bacteria are selected?

Answer 38

Expression of plasmid gene in bacteria (if bacterial promoter)

Question 39

What happens after expression of plasmid gene in bacteria?

Answer 39

Amplification of bacteria and purification of DNA for downstream uses - PCR, cloning, transfection into other cells or organisms

Question 40

What do all organisms do?

Answer 40

Read the same codons as the same amino acid

Question 41

What is AUG?

Answer 41

Methionine

Question 42

What is UGA?

Answer 42

Stop codon

Question 43

What is the significance of the universal genetic code?

Answer 43

We can transform a human gene into bacteria and it will still make the same protein

Question 44

What don't prokaryote genes have?

Answer 44

Introns

Question 45

What don't prokaryotes have?

Answer 45

The machinery to process eukaryotic introns (can't do splicing)

Question 46

What can be used when making recombinant proteins in bacteria?

Answer 46

Only the coding sequence

Question 47

What is used when making recombinant proteins?

Answer 47

The mRNA from the gene is copied and inserted into the plasmid as it doesn't have introns