Lecture 5

Question 1

What is gene cloning?

Answer 1

Cloning a single gene of interest and inserting it into a vector so that it can be copied quickly and its function and structure can be studied

Question 2

What is gene cloning not like?

Answer 2

It is not like somatic cell nuclear transfer (what everyone thinks of when people say cloning eg dolly the sheep)

Question 3

What is the first step when gene cloning?

Answer 3

Isolating the gene of interest from the chromosome in the host cell, this can be done using restriction enzymes or by using PCR

Question 4

What happpens with the gene once it has been isolated from the chromosome in gene cloning?

Answer 4

The fragment of DNA needs to be placed into a vector

This vector is usually a bacterial plasmid

Question 5

What does both the desired DNA fragment and the plasmid have to have been cut with to allow the joining of the two structures?

Answer 5

They both have to have been cut with the SAME restriction enzyme

Question 6

What happens to the plasmid after the gene has been inserted into it?

Answer 6

It is placed into a bacterial cell such as e.coli that can then reproduce and produce many of the bacterial cell containing the desired gene

Question 7

When can a gene be isolated using PCR?

Answer 7

If the gene has bee well studied and if it is under 5kb you can use PCR to amplify the DNA

Question 8

What can you use to isolate a specific gene that cannot be amplified via PCR?

Answer 8

Restriction enzymes are used to digest the genome and then individual segments are cloned and reassembled

Question 9

To act as a cloning vehicle what must something be able to do?

Answer 9

• The molecule must be capable of entering a host cell with the DNA molecule
• Once inside the cell they must be able to replicate and produce multiple copies of itself

Question 10

What are the 2 types of naturally occurring types of DNA molecule that can be used as vectors?

Answer 10

• Plasmids

- Bacteriophages

Question 11

What are plasmids and why are they useful during gene cloning?

Answer 11

• Extrachromosomal elements, double stranded and capable of replication
• Has an origin of replication and can multiply independent of the chromosome
• Easily isolated
• Easily manipulated
• High reproduction rate

Question 12

What are the basic properties that a plasmid MUST have?

Answer 12

• An origin of replication - it is required to replicate independently
• A dominant selectable marker - this is required so that when screening etc the bacteria carrying the desired plasmid can be distinguished
• Unique restriction endonuclease cleavage sites - essential to allow the insertion of foreign DNA fragments into the plasmid

Question 13

What happens when creating recombinant DNA molecules and what is it called?

Answer 13

It is called ligation

• Nucleases are used to cut, shorten or degrade nuclei acids
• Ligases are used to join nucleic acid molecules together

Question 14

Why are restriction endonucleases used during gene cloning?

Answer 14

• Because they are very precise and and produce reproducible results (they are used because each vector molecule has to be cut at exactly the same position on the circle)
• They cleave the vector at a single position opening up the circle so that new DNA can be inserted

Question 15

What do the restriction endonucleases and DNA lipase do when producing recombinant DNA?

Answer 15

• The restriction enzyme recognises a specific sequence in the DNA that is 4-8bp long
• On recognition of this site both strands of DNA become cleaved at that point because the restriction sites are short palandromic sequences
• The DNA is cut in a staggered manner leaving ‘sticky ends’ which can form H bonds with complementary sticky ends
• DNA ligase comes in and permanently binds the 2 complementary sticky ends together instead of them just being held by H bonds

Question 16

How many types of restriction enzyme are there and which is the most commonly used?

Answer 16

There are 4 types of restriction enzyme and they type II restriction enzyme is the most commonly used

Question 17

What can the palindromic sequence and the sticky ends also be referred to as?

Answer 17

• Axis of symmetry

- Two fold rotation of symmetry

Question 18

How does DNA ligase bind the two molecules together?

Answer 18

It creates phosphodiester bonds between them

Question 19

How is the recombinant plasmid inserted into the host cell?

Answer 19

• The bacterium and the plasmid are incubated together
• They are then chemically treated with CaCl2 which helps the transfer of the plasmid into the cell because the positively charged CaCl2 attracts the plasmid because of the negative charge on the DNA (the cell is now said to be a competent cell as it has the ability now to uptake the plasmid)
• The cells are then subjected to a heat shock which creates pores in the cell membrane allowing the plasmid to enter
• After the short heat shock the cells are subject to a low temperature to reseal the pores in the cell membrane (this cell is now said to be a transformed cell)

Question 20

How do we screen the cells to make sure that the plasmid has been taken up and the transformation has been successful?

Answer 20

• The plasmids that were chosen as a vector must have had a selective marker, which usually is an antibiotic resistance gene
• An experiment is done then with the cells on an agar plate treated with that specific antibiotic and any cells that haven’t taken up the new plasmid will NOT grow on the plate (you will only see bacteria grow that has successfully taken up the plasmid)

Question 21

How do we check that the ligation of the gene has been successful?

Answer 21

• We do this using another type of marker because the gene that is inserted into the plasmid is usually inserted into the middle of a second selective marker interrupting its function
• We can use the LacZ gene for blue-white screening (LacZ functions to break down lactose to glucose and galactose)
• The experiment to monitor this is to plate the cells alongside a chemical called X-Gal if the LacZ gene is still functioning then Beta galactosidase can break down X-Gal forming a deep blue colour - blue colonies therefore mean the plasmid is present but your gene isn’t
• A white colony will be present if the plasmid and the DNA have been successfully ligated