Lecture 18

Question 1

Why is gene cloning done?

Answer 1

Cloning is used to produce lots of identical copies of a particular gene or sequence of DNA in order to study the gene, sequence it or express it. Cloned genes allow us to determine the sequence of the gene (and protein), obtain leads into the function of the gene and manipulate the gene i.e mutations, insert the gene into cells or tissue of an organism and make large amounts of protein.

Question 2

What are some key definitions in cloning?

Answer 2

A clone is a large number of identical cells or molecules with a single ancestral cell or molecule, a DNA cloning vector is a carrier DNA molecule that allows attached DNA to be replicated in a cell e.g plasmids and viruses (SEE SLIDE).

Question 3

What are the two primary decisions in cloning a gene?

Answer 3

Decisions in cloning a gene include what vector to use (ease of use and size of DNA fragments), and what host to use (ease of use and correct production of genetic product).

Question 4

What are plasmids, how are they inherited and why are they good for DNA cloning?

Answer 4

Plasmids are a double stranded DNA molecule that replicates in cells independently of the host chromosome. They are inheriited by the daughter cells and are non essential for growth, they are ideal as cloning vectors because they are well characterised (sequence and function of genese known) and they are small, easy to purify and manipulate, they have na origin of replication, a selectable marker, unique restriction enzyme cleavage sites, easy methods to screen for recombinants and can have high copy numbers. Of these the origin of replication, a selectable marker and unique restriction enzyme cleavage sites are required.

Question 5

What does cloning with a piece of DNA and bacteria involve?

Answer 5

Cloning a piece of DNA with a plasmid and bacteria involves:

1. restriction, the plasmid vector and the fragment of interest must be digested with a restriction enzyme to produce sticky ends.
2. Ligation, the DNA fragment of interest must be ligated into the plasmid.
3. Introducing, adding the plasmid to the bacteria can be done through transformation (made competent by treating with ice cold CaCl2 and DNA and then heat shocked at 42 degrees) or electroporation, cells made competent to take up DNA by treatment with a large electric field.
4. Screening, in order to determine which cells ended up with the wanted combination. This could be done through checking for loss of antibiotic resistance by plating on specific antibiotics the plasmid should have if no insertion occurs e.g amp resistance but tet resistance only if no insertion occurs, plate on amp first, then again on amp and tet. Take colonies which don’t survive on tet. We could also screen for colour change e.g on PUC plasmid part of lacZ alpha gene is present but not all, meaning the alpha peptide is still needed and can be provided from a seperate gene, we can add Xgal to activate this enzyme and make the colonies which have no insert blue, this is known as alpha complementation.