DNA technology

Question 1

What are the 5 stages of making a protein using DNA technology and cloning?

Answer 1

1. Isolation of the DNA fragments that have the desired protein
2. Insertion of the DNA fragment into a vector
3. Transformation- the transfer of DNA into suitable host cells
4. Identification of the host cells that have successfully taken up the gene by use of gene markers
5. Growth/cloning of the population of host cells

Question 2

What is reverse transcriptase?

Answer 2

It is an enzyme that sythesises DNA from RNA and catalyses that reaction (RNA to DNA). Basically, it goes backwards to the usual DNA –> mRNA –> tRNA –> amino acid sequence.

Question 3

What does reverse transcriptase actually do?

Answer 3

It forms a copy of the requested gene from large amounts of relevant mRNA and then reverse transcriptase produces a cDNA strand which is complementary to the mRNA.
DNA polymerase then builds a double stranded DNA molecule on the template cDNA. And that is the copy.

Question 4

What is restriction endonuclease?

Answer 4

It is an enzyme that cut are used in bacteria to cut up viral DNA, therefore it comes in useful for slicing DNA up in to fragments. It cuts the DNA fragments out of the DNA with the same enzyme that cuts the plasmid open.
Restriction endonucleases only cut at a certain sequence and if it is the same enzyme cutting the DNA and the plasmid then their sticky ends will be complementary.

Question 5

Describe in vivo cloning.

Answer 5

1. DNA from the cell that produces the required protein and DNA in the plasmid are both cut by the same restriction endonuclease. (so they have complementary sticky ends).
2. The DNA fragments are then added to the open plasmids and mixed together with DNA ligase.
3. DNA ligase binds the DNA and the plasmids together.
4. Plasmids and the bacteria are mixed with calcium ions and the temperature is changed. These new conditions increase the permeability of the bacterial cells.

Question 6

How do you tell if the gene has been taken up by bacterial cells?

Answer 6

It may be resistant to an antibiotic.
It may make a fluorescent protein that is easily seen.
It may produce an enzyme whose action can be identified.