Recombinant DNA Technologies

Question 1

Recombinant DNA

Answer 1

Join sections of DNA together

Inserted into another organism to produce protein

Question 2

Manipulation of recombinant DNA

Answer 2

Plasmid
Restriction enzymes
DNA ligases
Transformation (amplify plasmids)

Question 3

Plasmid

Answer 3

Circular piece dsDNA
Origin of replication
Antibiotic resistance gene
Promoter

Question 4

Origin of replication

Answer 4

Initiation of replication using host DNA poly

Question 5

Antibiotic resistance gene

Answer 5

Selection of cells with plasmid

Question 6

Promoter

Answer 6

Drives expression of gene (with transcription factors)

Correct type with a certain type of cell used (ie. prokaryotic needs protomer used in prokaryotic cells)

Question 7

Restriction enzymes

Answer 7

Cut dsDNA at specific sequence

Question 8

DNA ligase

Answer 8

Phosphodiester to bond with DNA backbone via sticky ends

Question 9

Transformation and replication of plasmid

Answer 9

Transfer plasmid into bacteria - heat shock
Select bacteria by antibiotics
Bacteria replicate with plasmid

Question 10

Not using bacteria to produce recombinant DNA

Answer 10

Same but
3
Transfrom into bacteria (increase plasmid) then transfer into eukaryote

Question 11

Universal genetic code

Answer 11

All organisms “read” same genetic code

Can produce same protein with human gene in bacteria

Question 12

Problem with universal genetic code

Answer 12

Prokaryotic genes can’t process eukaryotic introns

Question 13

Steps to produce recombinant protein

Answer 13

1) Isolate
2) Clone plasmid
3) Transform bacteria
4) Grow cells with protein of interest
5) Purify

Question 14

Insulin gene of interest

Answer 14

cDNA = no introns
Only A & B held by disulfide bonds
Must be made in separate bacteria as pre-proprotein (doesn’t occur in bacteria)

Question 15

Insulin purify

Answer 15

IacZ = 2 selector (removed later)
Fuse protein
Cyanogen bromide cleave A&B
Mix = functional insulin

Question 16

Insulin in normal human

Answer 16

Preproinsulin - roughER -> proinsulin 3 disulfide bonds b/w a and b
proinsulin - golgi -> insulin c removed

Question 17

Sources of therapertic proteins

Answer 17

Non human
Prokaryates
Human

Question 18

Non-human therapeutic protein eg and problems

Answer 18

Pig, cows
E.coli
Immue response, incorrect structure = incorrect function

Question 19

Human therapeutic protein eg and problems

Answer 19

Blood
Potentially infectious (blood borne pathogens), low yield, tissue source

Question 20

Advantages of prokaryotic system

Answer 20

High yield
Low cost
Pathogen free

Question 21

Disadvantages of prokaryotic system

Answer 21

Proteins partially folded

Inability to form post-translational modifications

Question 22

Advantages of mammalian cells

Answer 22

Pre-proprotein

Processed efficiently and purification easier

Question 23

Disdvantages of mammalian cells

Answer 23

Expensive

Question 24

EPO

Answer 24

Chinese Hamster Ovary cells
Glycosylation = PTM
-> human and recombinant look different after glycoslation

Question 25

Pharming

Answer 25

Using whole animals

Question 26

Anti-thrombin

Answer 26

Decrease = spontaneous blood clots

Control AT production

Question 27

Anti-thrombin pharming??

Answer 27

Transgenic goat
Promoter that control gene ????
Milk specific promotor (easy, doesn’t harm goat)

Question 28

Anti-thrombin problem

Answer 28

Can’t use prokaryotes as PTMs essential (gamma – carboxylation)

Question 29

Designing new proteins

Answer 29

Create random mutation = regenerate PCR

Replicate in bacteria and analyse

Question 30

Gene therapy

Answer 30

Plasmid target specific cells using viral vectors

Stable/permanent as it intergrates into host cell chromosome

Question 31

Type 1 diabetes

Answer 31

Beta cells destroyed - autoimmune disorder

Plasmid + pre-proinsulin cDNA make liver cells insulin producing

Question 32

Gene editing

Answer 32

CRISPR-CAs