Recombinant DNA and cloning vectors

Question 1

What is a DNA vector?

Answer 1

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.)

Question 2

What is a vector containing foreign DNA termed?

Answer 2

Recombinant DNA

Question 3

What are the different recombinant vectors?

Answer 3

Plasmids
>circular dsDNA found in many but not all bacteria
>replicate independently of bacterial chromosomes

Bacterial Phages
>Lambda- bacterial phages
>used clinically to treat infectious diseases and kill bacteria

Viruses
>non-primate Lentiviruses- used to integrate DNA in mammalian cells
>Baculoviruses- used in combination with recombinant expression in insect cells

Question 4

Give some Disadvantage of using plasmids, bacterial phages and viruses as recombinant vectors.

Answer 4

unable to handle large pieces of DNA

> as a consequence we can use yeast artificial chromosomes (YACs) to introduce large pieces of DNA into a eukaryotic cell

Question 5

How do we utilise plasmid as a vector?

Answer 5

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

Question 6

Give some Features of Plasmid Vectors.

Answer 6

• 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)

Question 7

What happens if a large piece of DNA is inserted into plasmid?

Answer 7

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

Question 8

Describe the Process of inserting foreign piece of DNA into plasmid.

Answer 8

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

Question 9

How is antibiotic resistance achieved from recombinant DNA?

Answer 9

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

Question 10

How can you confirm if the recombinant DNA has been inserted into the right place in the right orientation?

Answer 10

by restriction mapping

Question 11

Give uses of Recombinant Vectors in the Clinic.

Answer 11

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

Question 12

Define biologics.

Answer 12

Recombinant protein drugs produced from living organisms (human, animal or microorganism)