Module 8 - Gene technologies

Question 1

What is recombinant DNA

Answer 1

Contains sections of DNA from 2 types of organisms

Question 2

In-vivo amplification

Explain isolation of desired gene

Answer 2

1. mRNA converted to cDNA using reverse transcriptase
2. cDNA single stranded is converted to a double strand with DNA polymerase
3. DNA from desired gene is cut with the restriction enzymes to form sticky ends
4. Hydrolysis reaction, restriction site is specific to enzyme, palindromic, 4-6 bases

Question 3

In-vivo amplification

What is a gene machine

Answer 3

Artificially joining small lengths of nucleotides

Question 4

In-vivo amplification

Explain insertion into vector

Answer 4

1. Vector carries DNA from one organism to another (e.g. bacteria or bacteriophage)
2. Same restriction enzyme used to cut plasmid
3. Sticky ends of plasmid will be complementary to the sticky ends of the desired gene- H bonds will form (A-T, C-G)
4. Join phosphodiester bonds with DNA ligase
5. Recombinant DNA is formed
6. Add promoter and terminator region
7. Add a marker gene (e.g. fluoresence or antibiotic resistance)

Question 5

In-vivo amplification

Explain transformation and identification

Answer 5

1. Return plasmid into bacteria- bacteria will become transgenic
2. E.g. calcium salts and heat shock, microinjection, electroporation, liposomes, conjugation etc.
3. Identify using the marker gene e.g. UV light to identify fluoresence or add antibiotic to kill bacteria that are not resistant

Question 6

2 marks

Recombinant DNA technology can involve the transfer of fragments of human DNA into bacteria. The bacteria are then used to produce human proteins.
Give two reasons why bacteria are able to use human DNA to produce human proteins.

Answer 6

1. The genetic/DNA code is universal
2. The mechanism of transcription is universal
3. The mechanism of translation is universal

Question 7

2 marks

Suggest and explain one reason why bacteria might not be able to produce every human protein.

Answer 7

1. Cannot splice pre-mRNA, so cannot remove introns
2. Do not have Golgi(apparatus, so cannot modify and package proteins
3. Do not have transcriptional factors required, so cannot carry out transcription/produce mRNA

Question 8

1 mark

Explain the purpose of the marker gene.

Answer 8

• Shows that the gene has been taken up by cells
• Shows transgenic/transformed cells
• Allows detection of genetically modified cells/organisms

Question 9

2 marks

Describe how the DNA is broken down into smaller fragments.

Answer 9

• Restriction ezymes
• Cuts DNA at specific base sequence/ brakes phosphodiester bonds

Question 10

2 marks

Describe the roles of two named types of enzymes used to insert DNA fragments into plasmids.

Answer 10

1. Restriction enzyme to cut plasmid
2. Ligase joins DNA to plasmid

Question 11

2 marks

Scientists manufactured large quantities of human insulin using genetic engineering.
They started by isolating mRNA from pancreas cells. From this they produced DNA which coded for insulin.
uggest two reasons why it was better to start with mRNA from pancreas cells rather than with the DNA from these cells.

Answer 11

1. Amount of mRNA > amount of DNA / multiple copies of mRNA
2. Insulin mRNA is found in pancreas cells
3. Introns removed in mRNA

Question 12

2 marks

Describe the role of restriction endonucleases in the formation of plasmids that contain donor DNA.

Answer 12

Cut donor DNA, to remove gene / length of DNA
Cut donor DNA and plasmid with the same enzyme that cuts at the same base sequence
Sticky ends single strand
Attachment complementary strand

Question 13

1mark

Describe the role of DNA ligase in the production of plasmids containing donor DNA.

Answer 13

Join/ anneal phosphodiester bonds

Question 14

2 marks

Some human DNA was cut into separate pieces using a restriction enzyme which produced a staggered cut. A scientist wanted to insert these pieces of DNA into plasmids and used the same restriction enzyme to cut the plasmids. Explain why the pieces of human DNA would be able to join to the cut DNA of the plasmids

Answer 14

• Sticky ends of plasmid is complementary to sticky ends of desired gene
• H bonds form between complementary base pairing A-T, C-G

Question 15

4 marks

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

Answer 15

1. Requires DNA fragment DNA polymerase, DNA nucleotides and primers
2. Heat to 95 °C to break hydrogen bonds and separate strands
3. Reduce temperature so primers bind to DNA/strands
4. Increase temperature, DNA polymerase joins nucleotides and repeat method

Question 16

2 marks

The polymerase chain reaction (PCR) can be used to obtain many copies of a particular gene.
Explain how the strands of DNA are separated during the PCR.

Answer 16

1. By heating
2. To break the H-bonds between complementary bases

Question 17

1 mark

In a particular PCR, two different primers are added to the DNA.
Why are primers required?

Answer 17

To allow the DNA polymerase to attach

Question 18

1 mark

In a particular PCR, two different primers are added to the DNA.
Suggest why two different primers are required.

Answer 18

Because the sequences at the ends of the target sequence are different / one is at the beginning and one at the end

Question 19

2 marks

Explain one way in which the polymerase chain reaction differs from DNA replication in a cell.

Answer 19

1. uses heat
2. to separate strands
   OR
3. PCR replicates pieces of DNA
4. because DNA has been cut
   OR
5. primer added in PCR
6. to initiate replication

Question 20

6 marks

The polymerase chain reaction (PCR) can be used to produce large quantities of DNA.
Describe how the PCR is carried out.

Answer 20

1 DNA heated to 95°C
2 strands separate
3 cooled / to temperature below 70°C
4 primers bind
5 nucleotides attach
6 by complementary base pairing
7 temperature 70 - 75°C
8 DNA polymerase joins nucleotides together
9 cycle repeated