In vitro + in vivo cloning

Question 1

What does ‘in vivo’ mean?

Answer 1

Inside a living organism

Question 2

What does ‘in vitro’ mean?

Answer 2

Outside of a living organism

Question 3

What is the promoter region? (in vivo)

Answer 3

Added at the start of the DNA fragment. This is a sequence of DNA which is the binding site for RNA polymerase to enable transcription to occur.

Question 4

What is the terminator region? (in vivo)

Answer 4

This is added to the end of the DNA fragment. It causes RNA polymerase to detach and stop transcription, so that only one gene at a time is copied into mRNA.

Question 5

How does the DNA fragment have to be modified to ensure transcription of these genes occurs?

Answer 5

You have to add a promoter and terminator region.

Question 6

What is a vector?

Answer 6

Something to carry the isolated DNA fragment into the host cell.

Question 7

What is the most commonly used vector in ‘in vivo’ cloning?

Answer 7

A plasmid

Question 8

What enzyme do you use to insert the DNA fragment into the plasmid?

Answer 8

Restriction endonuclease

Question 9

How does restriction endonuleases help you insert the DNA fragment into the plasmid?

Answer 9

You cut the plasmid with the same restriction endonucleases as the ones you used to cut the DNA fragment. This creates sticky ends which are complementary to the sticky ends on the DNA fragment.

Question 10

What enzyme is used to ‘glue’ the DNA fragment in place, inside the plasmid?

Answer 10

DNA ligase

Question 11

How does DNA ligase help insert the DNA fragment into the plasmid?

Answer 11

The DNA fragment and the ‘cut’ plasmid are combined and ligase sticks them together by catalysing the condensation reactions to form phosphodiester bonds between nucleotides.

Question 12

In short terms - what does DNA ligase do?

Answer 12

It joins the DNA/gene to the plasmid/vector

Question 13

In short terms, what do restriction endonucleases do?

Answer 13

They cut plasmid/vector

Question 14

What does ‘annealing’ mean?

Answer 14

Sticks two pieces of DNA together

Question 15

What is transformation? (in vivo)

Answer 15

Where the vector needs to be inserted into the host cell

Question 16

What needs to happen in order for the vector to be transformed into the host cell? (in vivo)

Answer 16

The membrane of the host cell needs to become more permeable

Question 17

How do you increase the permeability of the host cell’s membrane? What increases the fluidity of the cell membrane? (in vivo)

Answer 17

The host cells are mixed with Ca2+ and are heat shocked

Question 18

What does heat shocked mean? (in vivo)

Answer 18

A sudden increase in temperature

Question 19

What enables the vector to enter the cytoplasm of the host cell? (in vivo)

Answer 19

Increased permeability of membrane of host cell

Question 20

Why do you have to identify transformed cells? (in vivo)

Answer 20

Because not all of the host cells (most likely to be bacteria) will have taken up a recombinant plasmid

Question 21

Give three reasons as to why a host cell may not take up a recombinant plasmid: (in vivo)

Answer 21

The recombinant plasmid doesn’t get inside the cell.
The plasmid (that was cut) rejoins to itself before the DNA fragment entered
The DNA fragment sticks to itself rather than inserting itself into the plasmid

Question 22

What is a marker gene? (in vivo)

Answer 22

Genes on the plasmid that are used to identify which bacteria successfully took up the recombinant plasmid

Question 23

What are the three different marker genes that are used for identifying transformed cells? (in vivo)

Answer 23

Antibiotic resistance genes
Genes coding for fluorescent proteins
Genes coding for enzymes

Question 24

Describe how antibiotic-resistance markers are used to identify the transformed cells? (in vivo)

Answer 24

You insert a ‘tetracycline resistance’ gene and an ‘ampicillin resistance’ gene into a plasmid.
The you insert the DNA fragment for the target gene, into the tetracycline gene which disrupts it meaning that the gene will no longer create a functional proteins - it is no longer resistant to tetracycline
Then grow the bacteria on agar
Then use sterile velvet on a block to ‘stamp’ the master plate and transfer the colonies onto a plate with ampicillin antibiotic in the agar, leave for a couple of days
The colonies that grow must have had the plasmid inside of it due to the ampicillin resistance gene
Now use the sterile velvet block again, ‘stamp’ that plate and transfer to a plate with tetracycline antibiotic within the agar
The colonies that can still grow, must not contain recombinant plasmid
Look back at previous plate to identify which ones are missing and therefore the ones that contain the gene of interest.

Question 25

Describe how fluorescent markers are used to identify the transformed cells? (in vivo)

Answer 25

Jellyfish contain a gene which codes for a green fluorescent protein (GFP)
The GFP gene can be inserted into the bacterial plasmid
DNA fragment is then inserted into the middle of the GFP gene which disrupts it and prevents GFP production
Then grow the bacteria on agar and expose the colonies to UV light - the ones with the GFP gene still intact will grow, the ones with the DNA fragment within that gene will not be fluorescent.
ONLY NON-GLOWING COLONIES CONTAIN THE RE-COMBINANT PLASMID

Question 26

Describe how enzyme markers are used to identify the transformed cells? (in vivo)

Answer 26

Lactase can turn a certain substance from colourless to blue
The gene for this enzyme is inserted into the plasmid. The DNA fragment is then inserted into the middle of this gene - disrupting it, prevents lactase production
The bacteria are then grown on an agar plate with the colourless substance
The bacterial colonies which cannot turn this substance blue must contain the recombinant plasmid

Question 27

How do you grow the host cell? (in vivo)

Answer 27

A fermenter is used to grow multiple copies of the host cell which contains the recombinant plasmid - produces a large CLONED population of that bacterial cell

Question 28

What does PCR stand for?

Answer 28

Polymerase chain reaction

Question 29

What is the polymerase chain reaction used in?

Answer 29

In viTRO cloning

Question 30

What does PCR do? (short answer)

Answer 30

fragments of DNA can be amplified - in vitro

Question 31

Where does PCR take place?

Answer 31

In an automated machine

Question 32

What 5 pieces of equipment do you need to carry out PCR?

Answer 32

Thermocycler
DNA fragment to be amplified
DNA polymerase - taq polymerase
Primers
DNA nucleotides

Question 33

What is taq-polymerase?

Answer 33

It is a type of DNA polymerase obtained from bacteria which live in natural hot springs

Question 34

Why do we use taq-polymerase?

Answer 34

Because the enzyme has evolved to have a much higher optimum temperature than the usual DNA polymerase - optimum 72 degrees c and won’t denature until over 100 degrees c

Question 35

What is a primer?

Answer 35

A short sequence of single stranded DNA

Question 36

What must you ensure that a primer is?

Answer 36

The primer must be complementary to the ‘start’ and ‘end’ of the DNA fragment sequence that is being cloned

Question 37

Describe and explain how the PCR is used to amplify a DNA fragment: (4 marks) AQA EQ

Answer 37

Requires DNA fragment, DNA polymerase, DNA nucleotides, primers
Heat to 95 degreesC to break hydorgen bonds and separate strands
Redcue temperature so primers bind to DNA/strands
Increase temperature, DNA polymerase joins nucleotides (and repeat method)

Question 38

What three steps is the PCR made up of?

Answer 38

1. Denaturing
2. Annealing
3. Synthesis

Question 38

Describe how the polymerase chain reaction works?

Answer 38

Add in all of the ‘ingredients’
Increase the temperature to 95 to break the hydorgen bonds and split the DNA into single strands
The temperature is then decreased to 55 so that hydrogen bonds can form and attach the primers to the single DNA strands
DNA polymerase then attaches complementary free nucleotides and makes a new strand to align next to each template.
Temp is increased to 72 - taq optimum

Question 39

What are the three advantageous properties of PCR? And what are their advantages?

Answer 39

AUTOMATED - efficient
RAPID - 100 billion copies of DNA made within hours
DOESN’T REQUIRE LIVING CELLS - quicker and less complex techniques needed