Recombinant DNA technology Flashcards
(29 cards)
What are the four ways in which pharmaceuticals can be produced?
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.
How does genetic engineering and biotechnology work? What are the steps?
Isolation of gene for desired protein.
Cloning into a suitable vehicle.
Expression in a host cell system (typically mammalian, yeast or bacterial)
the introduction of a transgene has the potential to change the ______ of the organism
phenotype
What is the processing of insulin protein?
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
What does crb stand for and give the company name.
chain recombinant bacterial: A and B chains are synthesised separately. e.g. Genentech
What does prb stand for and give the company name.
proinsulin recombinant bacterial: Produce proinsulin and cleave central polypeptide enzymatically. e.g. Eli-Lilly
What are some advantages of recombinant insulin?
- cheap to manufacture
- very pure (98% pure)
- several modified forms have been produced with different release profiles.
Changes to which of the two chains causes fast acting release?
B
give an example of slow acting insulin
glargine
give examples of fast acting insulin
lispro
aspart
glulisine
Cloning is the process of
making genetically identical copies
How does gene cloning occur? Talk about the steps… cut, copy paste.
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.
How does E.coli prevent self destruction? By adding…
methyl groups
How are restriction endonucleases different to DNases?
Restriction endonucleases cut DNA at specific recognition sites.
Restriction enzymes e.g. EcoR1 carry:
- the name of their bacterial origin and
- a number
Briefly state how restriction endonucleases work.
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.
What are palindromes?
Sequences that read the same on opposite strands.
When restriction endonucleases do NOT make staggered cuts, but instead make a parallel cut, what are they called.
Blunt ends. These can also be used in molecular biology but they are not very good at sticking back.
Outline a few points about isolating cleaved DNA fragments and how it is carried out.
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.
Why are plasmids used as vectors and not chromosomes?
Chromosomes are large and not easy to manipulate
What are the different types of cloning vectors?
- plasmids (found in bacteria)
- cosmids (bigger plasmids)
- bacteriophages (viruses that infect bacteria)
- viruses
- bacterial artificial chromosomes (BACs)
- yeast artificial chromosomes (YACs)
Plasmids are
small, double stranded, self-replicating, extrachromosomal circular DNA molecules, distinct from the normal bacterial genome.
What are the four plasmid vector qualities?
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)
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!
to be cut at the same restriction site
hydrogen
DNA ligase
