In vivo and in vitro gene cloning

Question 1

What are the two methods of cloning the fragment of a gene that is useful

Answer 1

1) IN VIVO: This works by transferring the fragments to a host cell using a vector
2) IN VITRO: This works by using the polymerase chain reaction.

Question 2

Summarise why sticky ends are important

Answer 2

Because, provided the same restriction endonuclease is used, we can combine the DNA of one organism with that of any other organism.

Question 3

Explain why sticky ends are important

Answer 3

• If the same restriction endonuclease is used to cut DNA, then all the fragments produced will have ends complementary to one another as the endonuclease would cut the same specific recognition sequence.
• This means that the single stranded end of any one fragment can be joined to the single stranded end of any other fragment (they are ‘sticky’)
• Once the complementary bases of two sticky ends have paired up, an enzyme called DNA ligase is used to bind the phosphate-sugar framework of the two sections of DNA and so unite them as one.
• This means we can combine the DnA of any organism with that of any other organism.

Question 4

List the three stages of in vivo cloning

Answer 4

1. Preparing the DNA fragment for insertion
2. Insertion of the DNA fragment into a vector.
3. Introduction of DNA into host cells.

Question 5

Describe the role of the promoter region in the first stage of in vivo cloning: preparing the DNA fragment for insertion

Answer 5

• The preparation of the DNA fragment involves the addition of extra lengths of DNA.
• For the transcription of a gene to begin, the RNA polymerase must become attached to the promoter region of DNA.
• The nucleotide bases of the promoter attach both RNA polymerase and transcription factors and so begin the process of transcription.
• If we want the DNA fragment to transcribe mRNA in order to make a protein, it is essential that we attach it to the necessary promoter region to start the process.

Question 6

Describe the role of the terminator sequence in the first stage of in vivo cloning: preparing the DNA fragment for insertion

Answer 6

• The terminator sequence of DNA releases RNA polymerase and so ends transcription.
• Again, you need the terminator sequence added to the other end of the DNA fragment to stop transcription at the appropriate point.

Question 7

Describe the second stage of in vivo cloning: Insertion of DNA fragment into a vector

Answer 7

• Once an appropriate fragment of DNA has been cut from the rest of the DnA and the promoter and terminator regions added, it is joined into a carrying unit called a vector.
• This vector is used to transport the DNA into the host cell.
• There are different types of vector but the most common is bacterial plasmids.
• Plasmids almost always contain genes for antibiotic resistance and restriction endonucleases are used at one of these antibiotic resistant genes to break the plasmid loop.
• The restriction endonuclease used is the same one as was used to cut the DNA fragment which ensures that the sticky ends of the open end up plasmid are complementary to the sticky ends of the DNA sequence.
• When the DNA fragments are mixed with the opened up plasmids, they may be incorporated into them.
• Where they are incorporated, the join is made permanent using the enzyme DNA ligase.
• These plasmids are now recombinant DNA.

Question 8

Describe the third stage in in vivo cloning: Introduction of DNA into host cells

Answer 8

• Once the DNA has been incorporated into at least some of the plasmids, they must then be reintroduced into bacterial cells.
• This process is called transformation and involves the plasmids and bacterial cells being mixed together in a medium containing calcium ions.
• The calcium ions, and changes in temperature, make the bacterial membrane permeable, allowing the plasmids to pass through the cell-surface membrane into the cytoplasm.
• However, not all of the bacterial cells will possess the DNA fragments with the desired gene for the desired protein and the cells do not contain it are eliminated at this point through a variety of methods.

Question 9

What are the three reasons why not all the bacterial cells will posses the DNA fragments with the desired gene for the desired protein during the third stage of in vivo cloning

Answer 9

• Only a few bacterial cells (as few as 1%) take up the plasmids when the two are mixed together.
• Some plasmids will have closed up again without incorporating the DNA fragment.
• Sometimes the DNA fragment ends join together to form its own plasmid.

Question 10

Describe how we identify which bacterial cells have taken up the plasmid during in vivo cloning

Answer 10

• We use the fact that the plasmids contain genes for antibiotic resistance and this is unaffected by the introduction of the new gene.
• If the plasmids have a gene for, for example, ampicillin resistance the process is as follows:
• All the bacterial cells are grown on a medium that contains the antibiotic ampicillin.
• Bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance.
• These bacterial cells are able to break down ampicillin and therefore survive.
• The bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die.

Question 11

Why are further steps taken to identify recombinant bacterial cells after identifying which of the bacterial cells have taken up the plasmids

Answer 11

Some cells will have taken up the plasmids but not incorporated the new gene, and these cells will have also survived during the initial stage.

Question 12

List the ways of using marker genes to identify bacterial cells that may have taken up the plasmid but not incorporated the new gene

Answer 12

• Antibiotic resistant markers: replica plating
• Flourescent markers
• Enzyme markers

Question 13

How do you identify bacterial cells that may have taken up the plasmid but not incorporated the new gene

Answer 13

marker genes

Question 14

On a basic level how does using marker genes work

Answer 14

All marker genes are separate genes on the plasmid that are easily identifiable so tell us if the bacterial cell has incorporated this plasmids DNA into its own.

Question 15

Describe how antibiotic resistant marker genes are used to identify bacterial cells that have taken up the plasmids and the DNA fragment

Answer 15

• A technique called replica plating is used.
• This process uses the other antibiotic resistant gene in the plasmid: the gene that was cut in order to incorporate the required gene.
• As this gene has been cut, it will no longer produce the enzyme that breaks down the antibiotic.
• We can therefore identify these bacteria by growing them on a culture that contains the antibiotic.
• The problem with this method is that it destroys the cells that contains the required gene.
• However, by using replica plating you can identify colonies of bacteria containing the required gene.

Question 16

Describe how fluorescent marker genes can be used to identify. Which bacterial cells have taken up the plasmid

Answer 16

• In this more recent and rapid method, a gene from a jellyfish that produces a green fluorescent protein is transferred into the plasmid.
• The gene to be cloned is transplanted into the centre of the green fluorescent protein gene.
• Any bacterial cell that has taken up the plasmid with the gene that is to be cloned will not be able to produce the green fluorescent protein.
• bacterial cells that have not taken up the gene will continue to produce green fluorescent protein and fluoresce.

Question 17

Why is using fluorescent markers preferable top using antibiotic resistant markers to identify which bacterial cells have taken up the plasmid and incorporated the gene

Answer 17

• As the bacterial cells with the desired gene are not killed when using flourescent markers, there is no need for replica plating.
• Results can be obtained by simply viewing the cells under a microscope and retaining those that do not floouresce.
• This makes the process more rapid.

Question 18

Describe how enzyme markers can be used to see which bacterial cells have taken up the plasmid and incorporated its gene and which haven’t

Answer 18

• Another gene marker is the gene that produces the enzyme lactase which will turn a particular colourless substrate blue.
• The required gene is transplanted into the gene that makes lactase.
• If a plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactase.
• Therefore, when the bacterial cells are grown on the colourless substrate they will be unable to change its colour.
• Where the gene has not been taken up by the bacteria, they will turn the colourless substrate blue.
• These bacteria can be discounted.

Question 19

What is the polymerase chain reaction (PCR)

Answer 19

A method of copying fragments of DNA by in vitro cloning. The process is automated, making it rapid and effecient.

Question 20

List the things that the polymerase chain reaction (PCR) need for in vitro cloning

Answer 20

• The DNA fragment to be copied.
• DNA polymerase to join nucleotides
• Primers: short sequences of nucleotides that a set of bases complementary to those at one end of each of the two DNA fragments.
• Nucleotides which contain each of the four bases of DNA
• Thermocycler: a computer-controlled machine that varies temperature precisely over a period of time.

Question 21

List the three stages of the polymerase chain reaction (PCR)

Answer 21

1) Separation of the DNA strand
2) Addition (annealing) of the primers
3) Synthesis of DNA

Question 22

Describe the first stage of the polymerase chain reaction (PCR): separation of the DNA strand

Answer 22

• The DNA fragments, primers and DNA polymerase are placed in a vessel in a thermocycler.
• The temperature is raised to 95 degrees, causing the two strands of DNA to separate due to the breaking of the hydrogen bonds between the two DNA strands.

Question 23

Describe the second stage of the polymerase chain reaction: Addition (annealing) of the primers

Answer 23

• The mixture is cooled to 55 degrees, causing the primers to join (anneal) to their complementary bases at the end of the DNA fragment.
• The primers provide the starting sequences for DNA polymerase to begin DNA copying because DNA polymerase can only attach nucleotides to the end of an existing chain.
• Primers also prevent the two separate strands from simply rejoining.

Question 24

Describe the third stage of the polymerase chain reaction: synthesis of DNA

Answer 24

• The temperature is increased to 72 degrees.
• This is the optimum temperature for the DNA polymerase to add complementary nucleotides along each of the separated DNA strands.
• It begins at the primer on both strands and adds the nucleotides in sequence until it reaches the end of the chain.

Question 25

Why is the polymerase chain reaction (PCR) so quick

Answer 25

• Both separated strands are copied simultaneously so after one temperature cycle there are two copies of the original fragment.
• Once the two strands are completed, the process is repeated by subjecting them to the temperature cycle again, resulting in four strands.
• The whole temperature cycle takes two minutes- over 1 million copies of the original DNA can be made in 25 minutes and 100 billion copies can be manufactured in just a few hours.

Question 26

When is in vitro cloning especially useful

Answer 26

When a minute amount of DNA is available- eg. At the scene of a crime.

Question 27

What are the advantages of in vitro cloning

Answer 27

• It is extremely rapid- 100 billion copies of a gene can be produced in a matter of hours whereas this would take many days or weeks by in vivo cloning.
• It does not require living cells: all that is required is a base sequence of DNA that needs amplification.
• This means no complex culturing techniques, requiring time and effort, are needed.

Question 28

What is a disadvantage/ complicating factor of using PCR to clone

Answer 28

It also increases massively any other contaminating DNA present

Question 29

What are the advantages of in vivo gene cloning

Answer 29

• It is particularly useful where we wish to introduce a gene into another organism
• it involves almost no risk of contamination
• it is very accurate
• it cuts out specific genes
• it produces transformed bacteria that can be used to produce large quantities of gene products.

Question 30

Why is in vivo cloning particularly useful where we wish to introduce a gene into another organism

Answer 30

As it involves the use of vectors, once we have introduced the gene into a plasmid, this plasmid can be used to deliver the gene into another organism, such as a human. This technique is gene therapy.

Question 31

Why does in vivo cloning involve almost no risk of contamination

Answer 31

• Because a gene that has been cut by the same restriction endonuclease can match the sticky ends of the opened up plasmid.
• Contaminant DNA will therefore not be taken up by the plasmid.
• Conversely, in vitro cloning requires a very pure sample because any contaminant DNA will also be multiplied and could lead to a false result.

Question 32

Why is in vivo cloning very accurate

Answer 32

• The DNA copied has very few, if any, errors because mutations are very rare.
• However, in vitro cloning has errors in copying the DNA/contaminants and this will also be copies in subsequent cycles.

Question 33

Why is in vivo cloning cutting out specific genes an advantage

Answer 33

It is a precise procedure as the culturing of transformed bacteria produces many copies of a specific gene and not just copies of the whole DNA sample.