Lecture 19 - Recombinant DNA technology

Question 1

DIABETES

Answer 1

Type 1

Type 2

Question 2

NZ Type 1

Answer 2

1:5000

childhood onset

Question 3

NZ Type 2

Answer 3

15% lifelong risk

Question 4

What are recombinant DNA technologies?

Answer 4

Joining bits of DNA together (some from different species).

Insert into organism to produce (express) a useful protein.

Question 5

recombinant DNA technologies examples

Answer 5

Fluorescent jellyfish

Take DNA (green fluorescent protein) from jellyfish and place inside fly

Question 6

what are critical elements for recombinant DNA technologies?

Answer 6

Plasmids

Question 7

Plasmids

Answer 7

• circular pieces of double stranded DNA

• Replicate independently of
the host’s chromosomal
DNA

• Provide a benefit to hosts
e.g. antibiotic resistance

• Common in bacteria, but
also found in eukaryotes.

Question 8

Key components of recombinant DNA plasmids

Answer 8

Origin of replication (ORI)
Antibiotic resistance gene
Promoter

Question 9

Origin of replication (ORI)

Answer 9

allows initiation of replication using host DNA polymerase

Question 10

Antibiotic resistance gene

Answer 10

allows selection of cells containing plasmid.

Question 11

Promoter

Answer 11

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

Question 12

Promoter needs to

Answer 12

change to allow expression in.

Bacteria (prokaryote)
Fish (Eukaryote)
Neurons (cell type specific)

Question 13

Cutting and pasting DNA into plasmids

Answer 13

Restriction enzymes

Ligases

Question 14

Restriction enzymes

Answer 14

• found in bacteria – defense system to degrade foreign DNA

* Cut dsDNA at specific sequences

Question 15

DNA Ligases

Answer 15

• Complementary base pairing

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

Question 16

Amplifying plasmids

Answer 16

transformation

Question 17

Transformation

Answer 17

transfer of plasmids into bacteria

Question 18

Transformed bacteria selected by

Answer 18

antibiotic resistance contained on plasmid

Question 19

Amplification of bacteria and purification of DNA for downstream uses e.g.

Answer 19

PCR,

cloning, transfection into other cells or organisms

Question 20

Expression of plasmid gene in bacteria

Answer 20

if bacterial promoter

Question 21

The universal genetic code

Do All organisms ”read” the same codons as the same amino acids?

Answer 21

Yes

Question 22

AUG

Answer 22

methionine

Question 23

UGA

Answer 23

stop codon

Question 24

If we transform a human gene into bacteria will it still make the same protein?

Answer 24

Yes

Question 25

The catch when cloning eukaryotic genes for expression in prokaryotes

Answer 25

Use coding sequence only!

Question 26

Why do we Use coding sequence only when cloning eukaryotic genes for expression in prokaryotes?

Answer 26

Prokaryotic genes • Don’t have introns • don’t have machinery to process eukaryotic introns

Question 27

Recombinant DNA technologies combine

Answer 27

DNA from different species.

Question 28

Restriction enzymes and DNA ligase are used

Answer 28

to manipulate DNA and bacteria are used to amplify DNA

Question 29

A universal genetic code allows

Answer 29

genes from one species to be expressed in another species –