19

Question 1

I’ll

Answer 1

Oioin

Question 2

Type one diabetes - Frequency- onset

Answer 2

1:5000
- childhood onset (mostly)
- autoimmune - body attacks B cells in pancreas thus can’t produce insulin

Question 3

Both types if diabetes have

Answer 3

Genetic components to them

Question 4

Type 2 diabetes

Answer 4

• 15% lifelong risk
• lifestyle/ environment

Question 5

Diagram of type 1 diabetes

Answer 5

Question 6

Progression of gene therapy knowledge in 1960

Answer 6

1922: insulin first isolated from pig and cattle
1950s: identified protein sequences/structure

• treatment for diabetes was purified insulin for pigs and cattle

Question 7

Consequences (and why) of insulin purified from pancreas of cattle or pigs

Answer 7

• human amino acid sequence is similar but not identical
• immune responses range from local irritation to anaphylactic shock
• not always pure

(Cow insulin has 2 amino acid changes in alpha chain surrounding the disulphides bond, pig insulin has one extra amino acid at the C terminal of the peptide chain - the 3 amino acid changes in cow and one in pig at great enough to induce immune response if injected into human)

Question 8

Therapeutic proteins - human sources being used - pathogen transmission

Answer 8

• issues around safety (pathogen transmission), yield and source of protein (tissue availability)
• factors collected for haemophilic patients ending up containing HIV or hepatitis - couldn’t screen for HIV- many hemphouliacs ended up with aids
• growth hormone collected from cadavas used for dwarfism (need many pituitary glands to treat a single patient increasing risk every time you take a sample) this transmitted prion proteins - mad cow disease - Jacob

Question 9

Profession in gene therapy up to 2010

Answer 9

1922: insulin first isolated from pig and cattle
1950: insulin protein sequence ‘ structure identified
1973: recombinant DNA technology born
1977: insulin gene mapped too chromosome 11
1980: gene for insulin sequenced
1982: Eli Lilly produced the first recombinant insulin

Question 10

What are recombinant DNA technologies

Answer 10

Joining bits of DNA together (sometimes form different species). These are inserted onto an organisms to produce (express) a useful protein.

(E.g : Aequorea Victoria - GFP - produced fluorescent glow in stress response - can be inserted into other animals
- beta barrel structure )

Question 11

Plasmids are critical elements for…

Answer 11

… recombinant DNA technologies

Question 12

What are plasmids

Answer 12

• (usually) circular pieces of double stranded DNA
• replicate independently of the hosts chromosomal DNA

Question 13

Where are plasmids found

Answer 13

• common in bacteria
• also found in eukaryotes

Question 14

What do plasmids provide?

Answer 14

-provide a benefit to hosts EG antibiotic resistance

Question 15

Key components of recombinant DNA plasmids

Answer 15

• origin of replication
• antibiotic resistance
• promotor

Question 16

(In recombinant DNA plasmids) Origin of replication allows…

Answer 16

Initiation of replication using host DNA polymerase

Question 17

(In recombinant DNA plasmids) antibiotic resistance genes…

Answer 17

Allow selection of cells containing plasmid

Question 18

(In recombinant DNA plasmids) what does the promotor do?

Answer 18

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

Question 19

GFP (or variant) =

Answer 19

Gene

Question 20

Promotor allows

Answer 20

The expression of protein in a specific cell type or organism

Question 21

Promotor differ - can have promorotrs theta are only expressed in bacteria, eukaryotes or specific cell types

Answer 21

SAME GFP (or variants) but expressed in different cell types
- need a different promotor to allow different expression

Question 22

Where are restriction enzymes found and what do they do?

Answer 22

• naturally found in bacteria
• used as a defence system to degrade foreign DNA
• cut double stranded DNA at specific sequences

Question 23

Most common restriction enzyme an what sequence does it cut at (FIRST STEP OF CUTTING AND PASTING DNA INTO PLASMIDS)

Answer 23

EcoRI enxyme
Cuts at GAATTC sequence between the G and the A on the 5’ end and the G and the A on the 3’ end
- ends up with an overhang
- can insert this into other DNA with complementary ends

Question 24

The role of DNA ligase in cutting and pasting DNA into plasmids

Answer 24

• two DNAs have complementary base paring (DNA insert has a sticky end)
• DNA ligase catalyses the formation of the phosphodiester bone to repair nick in DNA backbone

Question 25

Process of amplifying plasmids - transformation

Answer 25

- transformation is the transfer of plasmids into bacteria’s - transformed bacteria selected by antibiotic resistance contained on plasmid - expression of plasmid gene in bacteria (if bacterial promotor) - amplification of bacteria and purification of DNA for down steam uses e.g. PCR, cloning, transfection into other cells or organisms (Yap page) - bacteria uses ORI to make many copies of the plasmid

Question 26

What is transformation

Answer 26

- the transfer of plasmids into bacteria

Question 27

Answer 27

Question 28

Two primary methods of artificial transformation …

Answer 28

- chemical transformation (requires chemically components cells uses cations to increase the permeability of the bacterium cell wall increasing the chance of DNA acquisition - includes heat shock step) - electroporaton ( involves the application of an electric field inducing holes in the cells perimeter allowing exogenous DNA to pass into cell)

Question 29

Universal genetic code is essential for taking these plasmids and expressing genes in other species

Answer 29

Yes

Question 30

The universal genetic code - what does it mean and what is its significance

Answer 30

- all organisms “read” the same codons as the same amino acids - AUG = methionine - UGA = stop codon - significance = we can transform a human gene into bacteria and it will still make the same protein

Question 31

Problems when cloning eukaryotic genes for expression in prokaryotes

Answer 31

- prokaryotic genes dont have in trials - prokaryotes dont have the machinery to process eukaryotic introns Instead: we take cDNA - take mature DNA thats already been spliced, isolate that and reverse transcribe it using reverse transcriptase making cDNA from the mRNA (thats already spliced) and we use that for recombinant DNA for the plasmid DO IT FOR ALL RECOMBINANT TECHNOLOGIES NOT JUST FOR BACTERIA as intervening sequences can be large

Question 32

Recombinant DNA technologies combine DNA from…

Answer 32

Different species

Question 33

Restriction enzymes and DNA ligase are used to

Answer 33

Manipulate DNA and bacteria and used to amplify DNA

Question 34

A universal genetic code allows genes from one species…

Answer 34

To be expressed in another species