In Vivo Cloning

Question 0

What are the two methods of cloning DNA fragments?

Answer 0

In vivo - by transferring the fragments to a host cell using a vector or in vitro - using the polymerase chain reaction

Question 1

Once the DNA fragments have been obtained, what is the next stage?

Answer 1

They must be cloned so that there is a sufficient quantity for medical or commercial use

Question 2

How are sticky ends joined after the DNA is cut by restriction endonucleases?

Answer 2

Using DNA ligase

Question 3

What are the sequences of DNA that are cut by restriction endonucleases called?

Answer 3

Recognition sites

Question 4

If the same restriction endonuclease is used to cut DNA, what will the fragments that are produced be?

Answer 4

Complementary to one another

Question 5

If the fragments that are produced are complementary, what does this mean?

Answer 5

It means that the single stranded end of any one fragment can be joined to the single stranded end of any other fragment

Question 6

Why are ‘sticky ends’ important, provided that the same restriction endonuclease is used?

Answer 6

We can combine the DNA of one organism with that of any other organism

Question 7

Once the DNA fragment has been cut from the rest of the DNA, what must it be joined into in order to be transported into the host cell?

Answer 7

A carrying unit, known as a vector

Question 8

What is the most commonly used vector?

Answer 8

Bacterial plasmids

Question 9

What do all plasmids tend to contain?

Answer 9

Genes for antibiotic resistance

Question 10

Where do restriction endonucleases break the plasmid loop?

Answer 10

At the antibiotic-resistance genes

Question 11

What restriction endonuclease is used to cut open the plasmid loop, and why?

Answer 11

The same one that was used to cut out the DNA fragment to ensure that the sticky ends of the opened up plasmid are complementary to the sticky ends of the plasmid

Question 12

What do the plasmids have once the DNA fragment is inserted into it?

Answer 12

Recombinant DNA

Question 13

What is the name of the process by which the modified plasmid is reintroduced into bacterial cells?

Answer 13

Transformation

Question 14

What does transformation involve?

Answer 14

The plasmids and bacterial cells are mixed together in a medium containing calcium ions

Question 15

What makes the bacteria permeable, allowing the plasmids to pass through the cell membrane and into the cytoplasm?

Answer 15

The calcium ions and changes in temperature

Question 16

Why will not all of the bacterial cells possess the DNA fragments?

Answer 16

Only a few of the bacterial cells take up the plasmids when the two are mixed together and some plasmids will have closed up again without incorporating the DNA fragment

Question 17

What does the task of finding out which bacterial cells have taken up the plasmid involve?

Answer 17

It involves using a gene for antibiotic resistance, which is unaffected by the introduction of the new gene

Question 18

Describe the process of using antibiotics to test for the uptake of a plasmid

Answer 18

All of the bacterial cells are grown on a medium that contains the antibiotic, the bacterial cells that have taken up the plasmids will have acquired the gene for antibiotic resistance, these bacterial cells are able to break down the antibiotic and therefore survive, the bacterial cells that have not taken up the plasmids will not be resistant to the antibiotic and therefore die

Question 19

What is the purpose of gene markers?

Answer 19

They are used to identify whether a gene has been taken up by bacterial cells by using a second, separate gene

Question 20

What makes the gene marker easily identifiable?

Answer 20

It may be resistant to an antibiotic, it may make a fluorescent protein that can be easily seen or it may produce an enzyme whose action can be identified

Question 21

What is the name of the process where those cells that have taken up the new gene are identified?

Answer 21

Replica plating

Question 22

What does replica plating involve the use of?

Answer 22

The other antibiotic resistance gene in the plasmid - the gene that was cut in order to incorporate the required gene

Question 23

How does replica plating allow the bacterial cells that contain the DNA fragment to be identified?

Answer 23

The antibiotic resistant gene that had been cut will no longer produce the enzyme that breaks down that antibiotic, in other words the bacteria that have taken up the required gene will no longer be resistant to that antibiotic

Question 24

What is the problem with replica plating?

Answer 24

The antibiotic that is used to identify the bacteria that contain the required gene will destroy those very cells

Question 25

How does the process of replica plating identify the bacteria that contain the modified plasmid if the antibiotic kills those cells?

Answer 25

The cells that survive from the treatment with the first antibiotic are cultured and placed in agar plates that contain genetically identical colonies of which a sample is transferred to a replica plate that contains the second antibiotic

Question 26

What is a more recent and more rapid method of identifying the successful uptake of a gene?

Answer 26

The transference of a gene from a jellyfish (GFP) which produces a green fluorescent protein into the plasmid

Question 27

What is another gene marker that allows you to visually identify the successfully modified bacteria?

Answer 27

The required gene can be transplanted into a gene that makes lactase - an enzyme that will turn a particular colourless substrate blue

Question 28

How does the use of enzyme markers such as lactase allow a successfully modified bacterial cell to be identified?

Answer 28

If the plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactase, therefore, the colourless substrate will not be turned blue