8.4 - Gene technologies

Question 1

Describe and explain how the polymerase chain reaction (PCR) is used to amplify a DNA fragment. (4)

Answer 1

• (Requires DNA fragment) DNA polymerase, (DNA) nucleotides and primers
• Heat to 95 °C to break hydrogen bonds (and separate strands);
  Accept temperature in range 90 to 95 °C
• Reduce temperature so primers bind to DNA/strands
  ‘accept temperature in range 40 to 65°C’
• Increase temperature, DNA polymerase joins nucleotides (and repeat method)

Question 2

The scientists used a radioactively labelled DNA probe to show that the cells of tobacco plant leaves contained the SUT1 gene. Describe how they would do this. (4)

Answer 2

• Extract DNA and add restriction endonucleases/restriction enzymes
• Separate fragments using electrophoresis
• (Treat DNA to) form single strands
  / (Treat DNA to) expose bases
  Ignore method used to separate strands
• The probe will bind to/hybridise/base pair with the SUT1/gene
• Use autoradiography (to show the bound probe)

Question 3

What is a DNA probe? (2)

Answer 3

• (Short) single strand of DNA
• Bases complementary (with DNA/allele/gene)

Question 4

Describe how the DNA is broken down into smaller fragments. (2)

Answer 4

• Restriction endonuclease/enzyme
• (Cuts DNA at specific) base sequence
  / (Breaks) phosphodiester bonds
  / (Cuts DNA) at recognition/restriction site

Question 5

The DNA on the nylon membrane is treated to form single strands. Explain why. (1)

Answer 5

• (So DNA) probe binds/attaches/anneals

Question 6

Explain the role of reverse transcriptase in RT-PCR. (1)

Answer 6

• Produces (c)DNA using (m)RNA

Question 7

Explain the role of DNA polymerase in RT-PCR. (1)

Answer 7

• Joins nucleotides to produce (complementary strand/s of) DNA

Question 8

Any DNA in the sample is hydrolysed by enzymes before the sample is added to the reaction mixture. Explain why. (2)

Answer 8

• To remove any DNA present
• As this DNA would be amplified / replicated

Question 9

Suggest one reason why DNA replication stops in the polymerase chain reaction. (1)

Answer 9

• Limited number of primers / nucleotides

Question 10

Limited number of primers / nucleotides. (1) del

Answer 10

• The same nitrogenous bases / triplets/codons code for the same amino acids in all living things/organisms

Question 11

Explain why restriction endonucleases are useful in genetic engineering? (2)

Answer 11

• They always cut DNA at the same place/restriction site/specific base sequence
• They generate sticky ends (when they cut DNA)
• Sticky ends (of the gene of interest and the plasmid vector) are complementary to each other

Question 12

Describe how reverse transcriptase enzymes can be used to produce cDNA. (2)

Answer 12

• Reverse transcriptase is mixed with mRNA (for the desired gene) AND free DNA nucleotides
• The reverse transcriptase uses the mRNA as a template and produces a new (single) strand of cDNA

Question 13

Describe how transformed cells can be identified. (3)

Answer 13

• The plasmid vector contains a marker gene
• The marker gene can code for antibiotic resistance/fluorescence
• Cells that survive exposure to antibiotics / fluoresce under UV light/GUS exposure have been transformed
  / cells that do not survive antibiotics exposure / do not fluoresce under UV light/GUS exposure have not been transformed

Question 14

Describe the process of genetic screening. (3)

Answer 14

• Patient DNA is isolated / the patient provides a sample of DNA
• The patient DNA is amplified using PCR
• Restriction endonucleases are used to cut the DNA into smaller fragments
• The fragments undergo gel electrophoresis
• DNA fragments are transferred to a nylon membrane
• DNA probes are added to the sample/nylon membrane
• The gel/sample is washed (to remove any unbound probe)
• UV light or an x-ray is used to reveal the position/presence of the probe

Question 15

Describe the method by which genetic fingerprinting is undertaken. (6)

Answer 15

• DNA is extracted from a sample (e.g. cheek cell)
• DNA is hydrolysed/cut into segments using restriction endonucleases
• Minisatellites / required core sequences must remain intact
• DNA fragments are separated using electrophoresis
• Explanation of electrophoresis e.g. mixture put into wells in gel and an electric current passed through
• Two strands of DNA separated OR gel is immersed in alkaline solution
• Perform southern blotting / cover with nylon / absorbent paper (to absorb DNA)
• DNA fixed to nylon/membrane using UV light
• Probe/radioactive marker added (which is picked up by required fragments) / probe/radioactive marker added that is complementary to minisatellites
• Areas with a probe attached are identified using X-ray film / autoradiography