Recombinant DNA technology

Question 1

What are the four ways in which pharmaceuticals can be produced?

Answer 1

1) Chemical synthesis: small molecules e.g. paracetamol
2) Direct extraction from biological source material: can extract blood, vaccines, antibodies, insulin etc from animal, plant or microbe
3) Expression using genetically engineered organisms: Recombinant therapeutic proteins can be expressed in bacteria, plant or animal cells e.g. monoclonal antibodies or recombinant insulin
4) A combination of approaches: chemical and biological entities can be combined, biological entities can be chemically or enzymatically modified.

Question 2

How does genetic engineering and biotechnology work? What are the steps?

Answer 2

Isolation of gene for desired protein.
Cloning into a suitable vehicle.
Expression in a host cell system (typically mammalian, yeast or bacterial)

Question 3

the introduction of a transgene has the potential to change the ______ of the organism

Answer 3

phenotype

Question 4

What is the processing of insulin protein?

Answer 4

Mature insulin molecule consists of two polypeptide chains (A; 21 aa and B; 30 aa) joined by disulphide bonds.
Signal sequence of precursor polypeptide (preproinsulin) is cleaved during transfer to ER.
Cleavage yields a second precursor (proinsulin; 108 aa).
Converted to insulin (51aa) by further proteolysis and removing the internal connecting polypeptide

Question 5

What does crb stand for and give the company name.

Answer 5

chain recombinant bacterial: A and B chains are synthesised separately. e.g. Genentech

Question 6

What does prb stand for and give the company name.

Answer 6

proinsulin recombinant bacterial: Produce proinsulin and cleave central polypeptide enzymatically. e.g. Eli-Lilly

Question 7

What are some advantages of recombinant insulin?

Answer 7

• cheap to manufacture
• very pure (98% pure)
• several modified forms have been produced with different release profiles.

Question 8

Changes to which of the two chains causes fast acting release?

Answer 8

B

Question 9

give an example of slow acting insulin

Answer 9

glargine

Question 10

give examples of fast acting insulin

Answer 10

lispro
aspart
glulisine

Question 11

Cloning is the process of

Answer 11

making genetically identical copies

Question 12

How does gene cloning occur? Talk about the steps… cut, copy paste.

Answer 12

Cut gene using restriction enzymes and isolate by gel electrophoresis.
Paste gene into suitable cloning vehicle/ vector (e.g. plasmid) using DNA ligase.
Introduce vector carrying the gene into host cell system e.g. E.coli, so that it can ‘COPY’ the gene and manufacture the protein.

Question 13

How does E.coli prevent self destruction? By adding…

Answer 13

methyl groups

Question 14

How are restriction endonucleases different to DNases?

Answer 14

Restriction endonucleases cut DNA at specific recognition sites.

Question 15

Restriction enzymes e.g. EcoR1 carry:

Answer 15

• the name of their bacterial origin and

- a number

Question 16

Briefly state how restriction endonucleases work.

Answer 16

Restriction endonucleases recognise molecular palindromes.
Most make staggered cuts in the sugar backbone of both DNA strands,
Gives rise to sticky ends.
These sticky ends can come back together and reform the sequence.

Question 17

What are palindromes?

Answer 17

Sequences that read the same on opposite strands.

Question 18

When restriction endonucleases do NOT make staggered cuts, but instead make a parallel cut, what are they called.

Answer 18

Blunt ends. These can also be used in molecular biology but they are not very good at sticking back.

Question 19

Outline a few points about isolating cleaved DNA fragments and how it is carried out.

Answer 19

We separate DNA using gel electrophoresis, according to their size by applying an electric field to the gel matrix (agarose or acrylamide).
We stain them with ethidium bromide in order to see them under UV light.
Once separated, DNA fragments can be cut out of the gel and used in ligase reactions.

Question 20

Why are plasmids used as vectors and not chromosomes?

Answer 20

Chromosomes are large and not easy to manipulate

Question 21

What are the different types of cloning vectors?

Answer 21

• plasmids (found in bacteria)
• cosmids (bigger plasmids)
• bacteriophages (viruses that infect bacteria)
• viruses
• bacterial artificial chromosomes (BACs)
• yeast artificial chromosomes (YACs)

Question 22

Plasmids are

Answer 22

small, double stranded, self-replicating, extrachromosomal circular DNA molecules, distinct from the normal bacterial genome.

Question 23

What are the four plasmid vector qualities?

Answer 23

1 - low molecular weight/ small size (less fragile/ easy to get into cells)
2 - high plasmid copy number (many copies of the gene to produce protein from)
3 - selectable ‘marker’ (allows us to select only those host cells that contain the plasmid)
4- unique restriction sites (for insertion of target gene in a single position)

Question 24

To insert a gene into a vector, both the gene and the vector need:
This produces matching sticky ends. These sticky ends will then join because of complementary base pairing through _______ bonds.
___ _____ seals the break in the sugar back bone.
This process depends on matching sticky ends “_____” each other in solution!

Answer 24

to be cut at the same restriction site
hydrogen
DNA ligase

Question 25

In order to copy our recombinant vector and produce protein from a gene, we need the machinery required for DNA replication, transcription and translation. This is provided by the;

Answer 25

host cell system/ expression host.

Question 26

Hosts tend to be:

Answer 26

fast growing cells such as bacterial (E.coli), yeast cells, mammalians or insect cell lines, but also transgenic plants and animals.

Question 27

What is the process by which we can get plasmids in E coli?

What are the three different ways in which this can be carried out?

Answer 27

Transformation
1 - chemical transformation/ heat shock (suspend cells in CacCl2 solution and jump temp to 42 and back to 37. Membrance becomes temporarily permeable to small, double stranded DNA)
2 - electrotransformation/ electroporation (put cells in cuvette and subject to a voltage spike of 200 to 200 volts for a few millisecs.
3 - Lipofection (encase DNA in liposomes and fuse with the cells)

Question 28

READ: BRIEF SUMMARY OF SELECTING RECOMBINANTS.

Answer 28

• Cut vector + gene of interest with matching restriction enzyme
• Ligate insert and vector together with DNA ligase
• Transform E.coli
• Plate on agar plates containing appropriate antibiotic (e.g ampicillin if pUC18)
• Only bacteria carrying plasmid will grow into colonies! (as they are ampicillin resistant)
• Pick a single colony (“a clone”)
• Grow in culture and extract protein!
• PROBLEM: SOMETIMES WE END UP WITH A RE-LIGATED VECTOR! WE END DUP WITH THE GENE WE STARTED WITH! (these get killed by the antibiotic)

Question 29

In reality, a ligation produces several types of molecules. Explain how we select recombinants using blue-white selection (lacZ) and the different possible outcomes.. (there’s three). State whether they grow on ampicillin or not.

Answer 29

1) Re-ligated vector (lacZ intact) –> growth on ampicillin; BLUE colonies
2) Recombinant vector (lacZ interrupted) –> growth on ampicillin; WHITE colonies a.k.a what we WANT to see!!!!
3) Re-ligated insert –> no growth on ampicillin