CGE: Amplifying DNA Fragments

Question 1

What must happen after the DNA fragment is isolated?

Answer 1

It must be amplified so you have sufficient quantity to work with.

Question 2

In vivo amplification

Answer 2

Copies of the DNA fragment are made inside of a host cell.

Question 3

What are the three steps of in vivo amplification?

Answer 3

1. DNA fragment is inserted into a vector creating recombinant DNA.
2. Vector transfers the DNA fragment into host cells - this tranforms the host cell.
3. Transformed host cells are identified and allowed to grow more.

Question 4

Describe first step of in vivo amplification:

Answer 4

DNA fragment inserted into vector.

1. Vector DNA is cut open using the same restriction endonuclease used to isolate the DNA containing target gene.
2. Sticky ends of vector are complementary to the sticky ends of the DNA fragment.
3. Vector DNA and DNA fragment are mixed with DNA ligase - ligation occurs and sticky ends join.
4. Recombinant DNA forms.

Question 5

DNA ligase

Answer 5

An enzyme that joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA, during in vivo amplification.

Question 6

Ligation

Answer 6

The process of the vecotr DNA and the DNA fragment joining, catalysed by DNA ligase.

Question 7

Describe the second step of in vivo amplification:

Answer 7

Vector transfers DNA fragment into host cell.

1. Plasmid vector - host cells must be persuaded to take it in.
2. Bacteriophage vector - host bacterium is injected with DNA and phage DNA intergrates with bacterial DNA.
3. Host cells are transformed.

Question 8

During in vivo amplification, what two types of vectors can be used?

Answer 8

Plasmids

Bacteriophages

Question 9

Bacteriophages

Answer 9

Viruses that infect bacteria.

Question 10

During in vivo amplification, how can host cells be persuaded to take in plasmid vectors and their DNA?

Answer 10

1. Host bacterial cells placed in ice-cold calcium chloride - makes cell walls more permeable.
2. Plasmids are added.
3. Mixture is heat-shocked for 1-2 mins.
4. Encourages cells to take in plasmids.

Question 11

Describe the third and final stage of in vivo amplification:

Answer 11

Identifying transformed host cells:

1. Transformed host cells contain target gene and markers that were inserted into vectors at the same time as target gene was.
2. Host cells are grown on agar plates and each creates a colony of cloned cells.
3. Transformed host cells will have colonies containing target gene and identifiable marker gene.
4. Transformed host cells are identified and allowed to grow more.

Question 12

What marker genes could be added to the vector during the first stage of in vivo amplification which would help transformed host cells be identified at a later stage?

Answer 12

1. Antibiotic resistance - host cells are grown on agar plates containing specific antibiotic, so only transformed host cells that have marker gene will survive and grow.
2. Fluorescence - when agar plate is placed under UV light only transformed cells will fluoresce.

Question 13

Roughly what percentage of host cells take up the vector during in vivo amplification?

Answer 13

5%

Question 14

What do you need if you want transformed host cells to produce the protein coded for by the DNA fragment?

Answer 14

The vector must contain the specific promoter and terminator regions.

Question 15

Promoter regions

Answer 15

DNA sequences that tell the enzyme RNA polymerase when to start producing mRNA.

Question 16

Terminator regions

Answer 16

Tell RNA polymerase when to stop producing mRNA

Question 17

What happens if you don’t have the right promoter region during in vivo amplification?

Answer 17

The DNA fragments won’t be transcribed by the host cell and a protein won’t be made.

Question 18

Promoter and terminator regions - how are they added to in vivo amplification?

Answer 18

May be present in the vector DNA or they may have to be added in along with the fragment.

Question 19

What are the two ways DNA can be amplified?

Answer 19

In vitro cloning

In vivo cloning

Question 20

In vitro cloning

Answer 20

A process of amplifying DNA whereby copies of the DNA fragments are made outside of a living organism using the polymerase chain reaction (PCR).

Question 21

How many copies of DNA fragments can be made in a certain time period?

Answer 21

Millions of copies in just a few hours.

Question 22

Describe the process of the PCR:

Answer 22

1. Mixture containing DNA sample, free nucleotides, primers and DNA polymerase is heated to 95^oC.
2. Breaks hydrogen bonds between DNA strands.
3. Mixture is cooled to 50-65^oC so primers can anneal to strands.
4. Reaction is heated to 72^oC so DNA polymerase can work.
5. DNA polymerase lines up free nucleotides with template strands - specific base pairing.
6. Two new copies of fragment DNA form - one cycle of PCR is complete.
7. Cycle starts again.

Question 23

During PCR, what temperatures are needed for the following stages:

• Initial heating of DNA mixture?
• Cooling to allow primers to anneal?
• Heating to allow DNA polymerase to work?

Answer 23

95^oC

50-65^oC

72^oC

Question 24

Primers

Answer 24

Short pieces of DNA that are complementary to bases at the start of the DNA fragment.

Question 25

DNA polymerase

Answer 25

An enzyme that creates new DNA strands.

Question 26

How does each PCR cycle affect the amount of DNA present?

Answer 26

Each PCR cycle doubles the amount of DNA.