B3. Amplifying DNA Fragments

Question 1

In vivo and in vitro gene cloning (describe each one)

Answer 1

In vivo cloning

• Where the gene copies are made within a living organism.
• As the organism grows and divides, it replicates the DNA, creating multiple copies of the gene

In vitro cloning

• where the gene copies are made outside of a living organism using the polymerase chain reaction (PCR)

Question 2

In vivo cloning

Once you’ve got the DNA fragment containing the target gene you can use it for in vivo cloning:

(3 Parts, 4,2,4)

Answer 2

Part 1 - Making recombinant DNA

1. The vector DNA is isolated.
2. The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene.
   This means that the sticky ends of the vector DNA 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. This process is called ligation.
4. The new combination of bases in the DNA (vector DNA + DNA fragment) is called recombinant DNA

Part 2 - Transforming cells

5. The vector with the recombinant DNA is used to transfer the gene into cells (called host cells).
6. Host cells that take up the vectors containing the gene of interest are said to be transformed.

If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA.

With a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting its DNA into it. The phage DNA (with the target gene in it) then integrates into the bacterial DNA

Part 3 - Identifying transformed cells
Only around 5% of host cells will take up the vector and its DNA, so it’s important to be able to identify which cells have been transformed. Marker genes can be used to identify the transformed cells

7. Marker genes can be inserted into vectors at the same time as the gene to be cloned. This means any transformed host cells will contain the gene to be cloned and the marker gene
8. Host cells are grown on agar plates and 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 gene and the marker gene.
9. The marker gene can code for antibiotic resistance-host cells are grown on agar plates containing the specific antibiotic, so only transformed cells that have the marker gene will survive and grow. Or the marker gene can code for fluorescence - when the agar plate is placed under a UV light only transformed cells will fluoresce
10. Identified transformed cells are allowed to grow more, producing lots and lots of copies of the cloned gene

Question 3

Tip: Bacteria can also be encouraged to take up DNA by ____________-the bacteria are given a very short electric shock that’s thought to create holes in the cell though which DNA can pass.

Answer 3

Tip: Bacteria can also be encouraged to take up DNA by electroporation-the bacteria are given a very short electric shock that’s thought to create holes in the cell though which DNA can pass.

Question 4

In-vivo cloning - Producing proteins

If you want the transformed host cells to produce the protein coded for by the DNA fragment, you need to make sure…

Promoter regions are…

Terminator regions tell it where to ______

Without the right promoter region…

Promoter and terminator regions may be present in the _________ ____ or they may have to be _______ in along with the _________

Answer 4

If you want the transformed host cells to produce the protein coded for by the DNA fragment, you need to make sure that the vector contains specific promoter and terminator regions.

Promoter regions are DNA sequences that tell the enzyme RNA polymerase where to start producing mRNA.

Terminator regions tell it where to stop.

Without the right promoter region, the DNA fragment won’t be transcribed by the host cell and a protein won’t be made.

Promoter and terminator regions may be present in the vector DNA or they may have to be added in along with the fragment

Question 5

In vitro cloning - DNA

This is where copies of the DNA fragments are made outside of a living organism using the polymerase chain reaction (PCR). PCR can be used to make millions of copies of a fragment of DNA in just a few hours. PCR has several stages and is repeated over and over to make lots of copies

(4 steps)

Answer 5

1. A reaction mixture is set up that contains the DNA sample, free nucleotides, primers and DNA polymerase.
   Primers are short pieces of DNA that are complementary to the bases at the start of the fragment you want.
   DNA polymerase is an enzyme that creates new DNA strands
2. The DNA mixture is heated to 95 °C to break the hydrogen bonds between the two strands of DNA. The mixture is then cooled to between 50 and 65 °C so that the primers can bind (anneal) to the strands
3. The reaction mixture is heated to 72 °C, so DNA polymerase can work. The 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.
4. Two new copies of the fragment of DNA are formed and one cycle of PCR is complete. The cycle starts again the mixture is heated to 95 °C and this time all four strands (two original and two new) are used as templates.

Each PCR cycle doubles the amount of DNA

Question 6

Tip: The DNA polymerase used in PCR is usually ___ ____________. It comes from bacteria that live in hot springs, so it is able to withstand high temperatures without denaturing. Most enzymes would denature well below 95 °C.

Answer 6

Tip: The DNA polymerase used in PCR is usually Taq polymerase. It comes from bacteria that live in hot springs, so it is able to withstand high temperatures without denaturing. Most enzymes would denature well below 95 °C.