Recombinant DNA and cloning vectors Flashcards
DNA Vector
DNA molecule used to carry foreign genetic material into another cell where it can be replicated and/or expressed (e.g. plasmid, Lambda phages etc.)
What is a vector containing foreign DNA termed?
recombinant DNA
Recombinant Vectors
Plasmids
- circular dsDNA found in many but not all bacteria
- replicate independently of bacterial chromosomes
- have a restricted host range
- are transferable by many means
Bacterial Phages
- Lambda- bacterial phages
- used clinically to treat infectious diseases
- transfer antimicrobial resistance through transduction
Viruses
- non-primate Lentiviruses - used to integrate DNA in mammalian cells
- Baculoviruses- used in combination with recombinant expression in insect cells
Artificial chromosomes
- yeast artificial chromosomes YACs: for introducing large segments of DNA
Disadvantage of using plasmids, bacterial phages and viruses as recombinant vectors
unable to hand large pieces of DNA
> as a consequence we can use yeast artificial chromosomes (YACs) to introduce large pieces of DNA into a eukaryotic cell
How do we utilise plasmid as a vector?
sometimes referred to as replicons
Plasmid reduced down to MINIMUM components necessary for the replication and maintenance of the vector within a bacterial organism:
- origin of replication
- other genes required for vector function and maintenance of that DNA piece
This structure can then be modified to introduce a foreign piece of DNA into plasmid and maintain that DNA as the plasmid/vector replicates or expresses a protein within the host cell
Features of Plasmid Vectors
- can be linearised at one or more sites in non-essential stretches of DNA
- can have DNA inserted into them
- can be re-circularised without the loss of the ability to replicate
- modified to replicate at high multiplicity (copy number) within a host cell
- contain selectable markers to select for them (e.g. antibiotic resistance marker)
- relatively small (4-5kb in size)
What happens if a large piece of DNA is inserted into plasmid?
plasmid becomes unstable and you get recombination, resulting in either:
- truncation
- cutting of gene of interest
- loss of plasmid altogether
*plasmid no longer viable/useful
Process of inserting foreign piece of DNA into plasmid
1) Multiple cloning site (piece of DNA) with different restriction enzyme sites is introduced to the plasmid
2) Restriction enzymes cut plasmid at specific restriction sites within a region of DNA non-essential to the maintenance of the plasmid. Circular DNA is now opened.
3) Foreign piece of DNA developed to have compatible restriction sites at the ends of its sequences.
4) Foreign piece of DNA inserted within the multiple cloning site by ligating (DNA ligase) both pieces of DNA together. Plasmid is now re-circularised
How is antibiotic resistance achieved from recombinant DNA?
Foreign DNA incorporated into plasmid to form vector:
1) vector transformed into E.Coli bacterial cells
2) vector contains antibiotic resistance gene, and would confer this resistance onto bacterial cells
3) Plate the transformed bacterial onto agar plate containing antibiotic, and only the recombinant E.Coli would grow
4) Isolate colonies, culture them, grow them and purify the protein
How can you confirm if the recombinant DNA has been inserted into the right place in the right orientation?
by restriction mapping
by restriction mapping
Recombinant Vectors in the Clinic
Recombinant vectors facilitate the production of recombinant protein drugs:
Human Insulin
-diabetes
Interferons (alpha and beta)
-viral hepatitis or MS
EPO
-kidney disease and anaemia
Factor XII
-haemophilia
tPA
-embolism, stroke
Biologics
Recombinant protein drugs produced from living organisms (human, animal or microorganism)
