3.8.4 Gene Technologies

Question 1

Describe the method of in-vivo
cloning with the use of an
antibiotic resistant marker
gene (8)

Answer 1

1. isolate wanted gene / DNA from another organism / mRNA from cell / organism;
2. using restriction endonuclease / restriction enzyme / reverse transcriptase to get DNA and
3. produce sticky ends;
4. use ligase to join wanted gene to plasmid;
5. include marker gene e.g. antibiotic resistance;
6. add plasmid to bacteria to grow (colonies)then (replica) plate onto medium where the marker gene is expressed;
7. not killed have antibiotic resistance gene and (probably) the wanted gene

Question 2

Describe the process of in-
vitro cloning using PCR (9)

Answer 2

1. DNA heated to 90 to 95°C;
2. strands separate;
3. cooled / to temperature below 70°C
4. primers bind; (primers identify the DNA sequence to be amplified)
5. nucleotides attach;
6. by complementary base pairing;
7. temperature 70 - 75°C;
8. DNA polymerase joins nucleotides together;
9. cycle repeated;

Question 3

Describe the use of DNA
probes in gene testing. (4)

Answer 3

1. probe will attach ( e.g. to allele);
2. attaches to one DNA strand;
3. as a result of complementary base pairing;
4. radioactivity detected on film / X-ray / by autoradiography;

Question 4

Outline the process of genetic
fingerprinting (10)5

Answer 4

1. DNA extracted from sample;
2. DNA cut into segments using restriction endonucleases;
3. Must leave VNTR / required core sequences intact;
4. DNA fragments separated using electrophoresis;
5. detail of process e.g. mixture put into wells on gel and electric current passed through;
6. immerse gel in alkaline solution / two strands of DNA separated;
7. Southern blotting / over with nylon / absorbent paper (to absorb DNA)
8. DNA fixed to nylon / membrane using UV light;
9. radioactive marker / probe added complementary to VNTR;
10. (areas with probe) identified using X-ray film