Amplifying DNA fragments

Question 1

What do you have to do once you’ve isolated your DNA fragments?

Answer 1

Amplify the DNA fragments so you have a sufficient quantity to work with

Question 2

What is in vivo cloning?

Answer 2

Cloning where copies of the DNA fragments are made inside living organisms

Question 3

Name the 3 steps of in vivo cloning?

Answer 3

1. The DNA fragment is inserted into the vector
2. The vector transfers the DNA fragments into host cells
3. Identifying transformed host cells

Question 4

What is a vector?

Answer 4

Something that is used to transfer DNA into a cell.
They can be plasmids or bacteriophages

Question 5

What are plasmids?

Answer 5

Small circular molecules of DNA in bacterium

Question 6

What are bacteriophages?

Answer 6

Viruses that infect bacteria

Question 7

Describe stage 1 of in vivo cloning
(the DNA fragment is inserted into a vector)

Answer 7

1. The DNA vector is inserted into vector DNA
2. The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA. The sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene
3. The vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA (ligation)

Question 8

Explain the process of ligation

Answer 8

DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector

Question 9

What is recombinant DNA?

Answer 9

The new combination of bases in the DNA

Question 10

Describe stage 2 of in vivo cloning
(the vector transfers the DNA fragment into host cells)

Answer 10

1. The vector with the recombinant DNA is used to transfer the gene into cells
2. If a plasmid vector is used host cells have to be persuaded to take in the plasmid vector and its DNA
3. If a bacteriophage vector is used the bacteriophage will infect the host bacterium by injecting its DNA into it. The phage DNA then integrates into the bacterial DNA.
4. Host cells that take up the vectors containing the gene of interest are said to be transformed.

Question 11

What are marker genes used for?

Answer 11

To identify the transformed cell

Question 12

Describe stage 3 of in vivo cloning
(identifying transformed host cells)

Answer 12

1. Marker genes can be inserted into vectors at the same time as the gene to be cloned which means any transformed host cells will contain the gene to be cloned and the marker gene.
2. Host cells are grown on agar plates. Each cell divides and replicates its DNA creating a colony of cloned cells. Transformed cells will produce colonies where all the cells contain the cloned cell and marker gene.
3. The marker gene can code for antibiotic resistance or fluorescence.
4. Identified transformed cells are allowed to grow more producing lots of copies of the cloned cell

Question 13

Why does the vector need to contain specific promoter and terminator regions?

Answer 13

To ensure the transformed host cells produce the protein coded for by the DNA fragment

Question 14

What are promoter regions?

Answer 14

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

Question 15

What happens if you do not have the correct promoter regions?

Answer 15

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

Question 16

What are terminator regions?

Answer 16

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

Question 17

What is in vitro cloning?

Answer 17

Cloning where copies of the DNA fragments are made outside of a living organism using the polymerase chain reaction (PCR)

Question 18

Explain the process of in vitro cloning

Answer 18

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

Question 19

What are primers?

Answer 19

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