Module 8 - Gene technologies

Question 1

What is recombinant DNA

Answer 1

Contains sections of DNA from 2 types of organisms

Question 2

Define recombinant DNA technology

Answer 2

Recombinant DNA technology involves the transfer of fragments of DNA from one organism, or species, to another

Question 3

In-vivo amplification

Explain isolation of desired gene

Answer 3

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 4

In-vivo amplification

What is a gene machine

Answer 4

Artificially joining small lengths of nucleotides

Question 5

In-vivo amplification

Explain insertion into vector

Answer 5

1. Vector carries DNA from one organism to another (e.g. plasmid 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 6

In-vivo amplification

Explain transformation and identification

Answer 6

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 7

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 7

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

Question 8

2 marks

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

Answer 8

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 9

1 mark

Explain the purpose of the marker gene.

Answer 9

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

Question 10

2 marks

Describe how the DNA is broken down into smaller fragments.

Answer 10

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

Question 11

2 marks

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

Answer 11

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

Question 12

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.
Suggest two reasons why it was better to start with mRNA from pancreas cells rather than with the DNA from these cells.

Answer 12

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 13

3 marks

Use your knowledge of enzymes to explain why restriction enzymes only cut DNA at specific restriction sites.

Answer 13

1. Different lengths of DNA have different base sequences / cut at specific sequence
2. Results in different shape / different shape of active site
3. Therefore specific sequence will only fit active site of enzyme

Question 14

4 marks

Cancer drugs usually interfere with DNA replication. Use this information to explain why the cancer drugs are administered as prodrugs and not the active form.

Answer 14

1. All cells contain DNA
2. Would stop DNA replication in normal cells
3. Stops cell division
4. Named example on growth / repair e.g. no new blood cells made / no wound healing

Question 15

2 marks

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

Answer 15

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 16

1mark

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

Answer 16

Join/ anneal phosphodiester bonds

Question 17

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 17

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

Question 18

4 marks

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

Answer 18

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 19

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 19

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

Question 20

1 mark

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

Answer 20

To allow the DNA polymerase to attach

Question 21

1 mark

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

Answer 21

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

Question 22

2 marks

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

Answer 22

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 23

6 marks

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

Answer 23

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

Question 24

BHA version

PCR- In-vitro amplification

Answer 24

• Add DNA nucleotides, tAq DNA polymerase and primers
• Primer:
  1. Short section of single stranded DNA
  2. Initiates replication.
  3. Marks beginning and ends of the part of DNA needed for attachment of enzymes or nucleotides
  4. Keep strands apart.
• TAq DNA polymerase is thermostable- high temp does not break hydrogen/ ionic/ disulfide bonds
• Heat to 95C to break H bonds and seperate strands
• Cool to 65C to allow primer to bind and DNA nucleotides to attach by complementary base pairs (A-T, C-G)
• Heat to 72C to anneal phosphodiester bonds- optimum temp for DNA polymerase
• Repeat cycle many times. Total DNA- start value x 2^n (n- number of divisions)

Question 25

# 1 mark Explain the role of reverse transcriptase in PCR

Answer 25

Converts mRNA to cDNA

Question 26

# 1 mark Explain the role of DNA polymerase in PCR

Answer 26

Joins nucleotides to produce DNA

Question 27

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

Answer 27

1. To remove any DNA present 2. As this DNA would be amplified

Question 28

# 1 mark Suggest one reason why DNA replication stops in the polymerase chain reaction.

Answer 28

Limited number of primers/nucleotides

Question 29

# 2 marks Scientists have used the RT-PCR method to detect the presence of different RNA viruses in patients suffering from respiratory diseases. The scientists produced a variety of primers for this procedure. Explain why.

Answer 29

1. Base sequences differ 2. Different complementary primers required

Question 30

# 1 mark In the polymerase chain reaction, DNA is heated to 95 °C and nucleotides, enzymes and DNA primers are added to the mixture. Explain why the DNA is heated to 95 °C.

Answer 30

1. to separate the two strands of the DNA 2. to break the hydrogen bonds

Question 31

# 1 mark What are DNA primers?

Answer 31

short section of single stranded DNA

Question 32

# 1 mark Why are DNA primers added during the polymerase chain reaction?

Answer 32

1. to mark beginning and ends of the part of DNA needed for attachment of enzymes or nucleotides 2. Keep strands seperate

Question 33

# 1 mark What is the advantage of the enzyme used in the polymerase chain reaction being thermostable?

Answer 33

1. would not be denatured 2. must be heated to 95 °C / must withstand high temps

Question 34

# 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 34

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

Question 35

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

Answer 35

Cannot splice pre-mRNA, so cannot remove introns Do not have Golgi apparatus, so cannot process/modify proteins Do not have transcriptional factors required, so cannot carry out transcription/produce mRNA

Question 36

# 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 36

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

Question 37

# 1 mark Suggest and explain one reason why bacteria might not be able to produce every human protein. | bacteria- prokaryotes, human- eukaryotes

Answer 37

Cannot splice pre-mRNA, so cannot remove introns (no intron splicing in prokaryotes) OR Do not have Golgi apparatus, so cannot process/modify proteins OR Do not have transcriptional factors required, so cannot carry out transcription/produce mRNA

Question 38

# 5 marks Plasmids can be modified by genetic engineering and inserted into bacteria. These bacteria can then make useful substances normally made by another organism. Explain how modified plasmids are made by genetic engineering and how the use of markers enable bacteria containing these plasmids to be detected.

Answer 38

1. isolate desired gene from another organism 1. using restriction enzyme and produce sticky ends 1. use ligase to join wanted gene to plasmid 1. also include marker gene e.g. antibiotic resistance 1. add plasmid to bacteria to grow colonies then replica plate onto medium where the marker gene is expressed 1. bacteria / colonies not killed have antibiotic resistance gene and the desired gene

Question 39

# 2 marks Explain why PCR is used during genetic fingerprinting

Answer 39

1. Only small amounts of DNA obtained- PCR increases mass of DNA 2. So enough DNA available for genetic fingerprinting

Question 40

# 2 marks What is the advantage of DNA taq polymerase being thermostable

Answer 40

1. Wouldnt be denatured 2. Must be heated to 95C/ withstand high temp | High temp wont break H/ ionic/ disulfide bonds