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BIOC192 > Lecture 19: RECOMBINANT DNA TECHNOLOGIES > Flashcards

Lecture 19: RECOMBINANT DNA TECHNOLOGIES Flashcards

1
Q

How many people get type 1 diabetes?

A

1 in 5000

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2
Q

When does type 1 diabetes develop?

A

Childhood usually

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3
Q

What is type 1 diabetes?

A

Loss of beta cells in the pancreas which make insulin

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4
Q

How many people get type 2 diabetes?

A

15%

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5
Q

When does type 2 diabetes develop?

A

Lifelong risk

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6
Q

What is type 2 diabetes?

A

Resistance to insulin

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7
Q

What happens in 1922?

A

Insulin first isolated from pig and cattle pancreas

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8
Q

What happened in the 1950’s?

A

Insulin proteins sequence/structure identified

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9
Q

What happened in 1973?

A

Recombinant DNA technology introduced (PCR and restriction enzymes)

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10
Q

What happened in 1977?

A

Insulin gene mapped to chromosome 11

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11
Q

What happened in 1980?

A

Gene for insulin sequenced

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12
Q

What happened in 1982?

A

Eli Lilly produced first recombinant insulin

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13
Q

Where was insulin purified from?

A

The pancreas of cattle or pigs

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14
Q

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

A

The amino acid sequences are similar but not identical

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15
Q

What is the structure of insulin?

A

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

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16
Q

How many differences between human and cow insulin?

A

2

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17
Q

How many differences between human and pig insulin?

A

1

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18
Q

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

A

Immune responses range from local irritation to anaphylactic shock

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19
Q

What is the problem with cow and pig insulin?

A

It isn’t always 100% pure

20
Q

What has also been used as therapeutic proteins?

A

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

21
Q

What are recombinant DNA technologies?

A

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

22
Q

What is the structure of plasmids?

A

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

23
Q

How do plasmids replicate?

A

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

24
Q

Where are plasmids found?

A

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

25
Q

What do plasmids provide?

A

A benefit to hosts - antibiotic resistance

26
Q

What are the key components of recombinant DNA using plasmids?

A

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

27
Q

What does the ORI allow?

A

Initiation of replication using host DNA polymerase

28
Q

What does the antibiotic resistance gene allow?

A

Selection of cells containing plasmid

29
Q

What does the promoter do?

A

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

30
Q

What must the promoter do?

A

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

31
Q

What is used to cut DNA?

A

Restriction enzymes

32
Q

Where are restriction enzymes found?

A

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

33
Q

What do restriction enzymes do?

A

Cut the double stranded DNA of the plasmid at specific sequences

34
Q

How is the DNA insert made?

A

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

35
Q

What does DNA Ligase do?

A

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

36
Q

What is transformation?

A

Transfer of plasmids into bacteria

37
Q

What are transformed bacteria selected by?

A

Antibiotic resistance contained on plasmid

38
Q

What happens after the transformed bacteria are selected?

A

Expression of plasmid gene in bacteria (if bacterial promoter)

39
Q

What happens after expression of plasmid gene in bacteria?

A

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

40
Q

What do all organisms do?

A

Read the same codons as the same amino acid

41
Q

What is AUG?

A

Methionine

42
Q

What is UGA?

A

Stop codon

43
Q

What is the significance of the universal genetic code?

A

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

44
Q

What don’t prokaryote genes have?

45
Q

What don’t prokaryotes have?

A

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

46
Q

What can be used when making recombinant proteins in bacteria?

A

Only the coding sequence

47
Q

What is used when making recombinant proteins?

A

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

BIOC192 (38 decks)

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