Gene Technology- Amplifying DNA Fragments

Question 1

What is a way of amplifying your DNA fragments?

Answer 1

Using in vivo gene cloning

Question 2

What is a vector?

Answer 2

Something that’s used to transfer DNA into a cell

Question 3

How is the vector DNA cut open?

Answer 3

Using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene

Question 4

Why is the same restriction endonuclease used?

Answer 4

So the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene

Question 5

What is the role of DNA ligase?

Answer 5

Joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA- ligation

Question 6

What is recombinant DNA?

Answer 6

The new combination of bases in the DNA (vector DNA + DNA fragment)

Question 7

What is the vector with the recombinant DNA used for?

Answer 7

To transfer the gene into cells (host cells)

Question 8

What is the role of marker genes?

Answer 8

To identify transformed cells by being inserted into vectors at the same time as the gene to be cloned which means that any transformed host cells will contain the gene to be cloned and the marker gene

Question 9

What is a transformed cell?

Answer 9

Host cell that takes up the vector

Question 10

Where are host cells grown?

Answer 10

On agar plates

Question 11

How do host cells create a colony of cloned cells?

Answer 11

Each cell divides and replicates its DNA

Question 12

What colonies do transformed cells produce?

Answer 12

Where all cells contain the cloned gene and the marker gene

Question 13

What can the marker gene code for?

Answer 13

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

Question 14

What are promoter regions?

Answer 14

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

Question 15

What are terminator regions?

Answer 15

DNA sequences that tell RNA polymerase when to stop producing mRNA

Question 16

What happens without the right promoter region?

Answer 16

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

Question 17

What does in vivo mean?

Answer 17

Inside the body

Question 18

What does in vitro mean?

Answer 18

Outside the body

Question 19

What does in vitro cloning use?

Answer 19

Polymerase chain reaction (PCR)

Question 20

What can PCR be used for?

Answer 20

To make millions of copies of a DNA fragment in just a few hours

Question 21

What does the reaction mixture that is set up during in vitro cloning contain?

Answer 21

The DNA sample, free nucleotides, primers and DNA polymerase

Question 22

What are primers?

Answer 22

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

Question 23

What is DNA polymerase?

Answer 23

An enzyme that creates new DNA strands

Question 24

What temperature is the DNA mixture heated to in order to break the hydrogen bonds between the 2 DNA strands?

Answer 24

95 degrees C

Question 25

Why is the mixture cooled to between 50 and 65 degrees C after the hydrogen bonds are broken?

Answer 25

So that the primers can bind (anneal) to the strands

Question 26

What temperature is the DNA mixture heated to in order for DNA polymerase to work?

Answer 26

72 degrees C

Question 27

What does the DNA polymerase do?

Answer 27

Lines up free DNA nucleotides alongside each template strand and joins the nucleotides together. Specific base pairing means new complementary strands are formed

Question 28

What is the product when one cycle of PCR is complete?

Answer 28

Two new copies of the fragment of DNA are formed

Question 29

What happens after one cycle of PCR is complete?

Answer 29

The cycle starts again, with the mixture being heated to 95 degrees C and this time all four strands are used as templates

Question 30

What does each PCR cycle do to the amount of DNA?

Answer 30

Doubles the amount

Question 31

What is the whole process of PCR?

Answer 31

1. A reaction mixture is set up, containing the DNA sample, free nucleotides, primers and DNA polymerase
2. DNA mixture is heated to 95 degrees C to break the hydrogen bonds between the two DNA strands
3. Mixture is the cooled to between 50 and 65 degrees C so that the primers can bind (anneal) to the strands
4. Reaction mixture is heated to 72 degrees C so DNA polymerase can work
5. DNA polymerase lines up free DNA nucleotides alongside each template strand and joins the nucleotides together- specific base pairing means new complementary strands are formed
6. Two new copies of the DNA fragment are formed and one cycle of PCR is complete
7. Cycle starts again, with mixture being heated to 95 degrees C and this time all four strands are used as templates
8. Each PCR cycle doubles the amount of DNA