3.8.4.1 Recombinant Technology

Question 1

What does recombinant DNA. technology involve

Answer 1

Transfer of fragments of DNA from one organism, or species, to another

Question 2

What is genetically modified organisms called

Answer 2

Transgenic

Question 3

How to make DNA fragments

Answer 3

Conversion of mRNA to complementary DNA (cDNA), using
reverse transcriptase

Using restriction enzymes to cut a fragment containing the desired gene from DNA

Creating the gene in a ‘gene machine’.

Question 4

How to make DNA fragments using restriction enzymes

Answer 4

Restriction enzyme cuts DNA, creating sticky ends.

Cut double stranded DNA by hydrolysing the phosphodiester backbone.

Futher enzymes extract the gene from the longer piece of cut DNA.

Question 5

What are restriction enzymes

Answer 5

Enzymes that cut double-stranded DNA at specific recognition sequences

Question 6

What are sticky ends

Answer 6

Overhangs of single-stranded DNA at the ends of the fragment

Question 7

How to make DNA fragments using reverse transcriptase

Answer 7

mRNA is reverse-transcribed into complementary DNA (cDNA) using reverse transcriptase.

The extracted mRNA is mixed with DNA nucleotides and reverse transcriptase.

mRNA used as a template to synthesise the complementary DNA

Question 8

How to make DNA fragments using a gene machine

Answer 8

DNA fragments can be synthesised from scratch by fixing a nucleotide onto a solid support.

Nucleotides are individually added in the correct order.

Protecting groups are added to prevent branching of the nucleotide chain.

Oligonucleotides are synthesised and joined together.

Question 9

What are the two ways of amlifying DNA fragments?

Answer 9

In vitro

In vivo

Question 10

Example of in vitro method of amplifying DNA fragments

Answer 10

Polymerase Chain Reaction (PCR)

Question 11

What is required in a PCR mixture?

Answer 11

DNA sample

Free DNA nucleotides

Primers (short DNA that bind initially and initiate replication)

DNA polymerase (from a thermophilic)

Question 12

Steps of PCR

Answer 12

Separation of the DNA strands - heated to 95C which causes hydrogen bonds between the DNA strands to break.

Annealing of the primer - cooled to around 60C which allows the primer to anneal to the DNA.

DNA synthesis - temperature increased to 72C. DNA polymerase catalyses the formation of phosphodiester bonds between the free DNA nucleotides using complementary base pairing rules.

Question 13

Example of in vivo method of amplifying DNA fragments

Answer 13

Transforming host cells

Question 14

How is a vector created for in vivo DNA amplifying

Answer 14

Plasmid is cut using the same restriction enzymes that we used to extract the gene, creating complimentary sticky ends.

Promoter and terminator regions added to the gene.

Fragment inserted into plasid.

DNA ligase anneals complientary sticky ends.

Recombinant DNA produced.

Question 15

What is the promoter and terminater used for in in vivo?

Answer 15

Promoter: Tells RNA polymerase where to start transcribing mRNA

Terminator: Tells RNA polymerase when to stop

Question 16

How is recombinant DNA inserted into a host cell?

Answer 16

Plasmid is mixed with bacterial cells using heat shock at 42C. Increasing membrane permeability.

All cells taking up the recombinant DNA are transformed.

Question 17

How are transformed cells located?

Answer 17

A radioactive or fluroscent marker gene

Question 18

What are in vivo technqiues disadvantaged?

Answer 18

Take longer.

Not all cells will become transformed.

Question 19

Evaluate DNA technology effect on animals

Answer 19

Positive:
Reduces cost
Increases production to meet supply

Negative:
Animals are sentiant
Health implications and harm
Reduces population variation

Question 20

Evaluate DNA technology effect on plants

Answer 20

Positive:
Prevents famines due to pathogens
Reduces cost
Increases production

Negative:
Impacts traditional farming
Potential harm to human health from food
Plants can easily interbreed

Question 21

Evaluate DNA technology effects on bacteria

Answer 21

Positive:
Less ethical concerns
Bacteria swap DNA amongst plasmids
Can be contained in the labrotary without interbreeding

Negative:
Potential contamination
Antibiotic resistance genes could create untreatable pathogens

Question 22

What is somatic gene therapy?

Answer 22

Certain types of cells are altered in their genome which makes them function. Will not be inherited and will need to be repeatedly done.

Question 23

What is germinline therapy?

Answer 23

Alters the gamete or zygote so changed the genome of the organism and potential offspring.

Could pose risks to other genes, cancer or complications.

Question 24

Describe how restriction endonuclease and DNA ligase are used to insert a gene into a plasmid [2]

Answer 24

[1] Restriction endonucleases/enzymes cuts plasmid OR produce sticky ends

[2] Ligase joins ‘sticky ends’ OR forms phosphodiester bonds

Question 25

Explain the importance of knowing base sequences in PCR [2]

Answer 25

[1] For primers [2] (To produce) a complementary base sequence

Question 26

Describe how DNA could be screened for all harmful mutations in a gene [4]

Answer 26

[1] Use of PCR to amplify (DNA sample); [2] Cut (DNA) using restriction endonuclease/enzymes [3] Separate (DNA fragments) using electrophoresis [4] Addition of (labelled) DNA probes and binding [5] (Mutations) identified by fluorescence/radioactivity

Question 27

Give 2 reasons why bacteria are able to use human DNA to produce human proteins [2]

Answer 27

[1] genetic/DNA code is universal [2] (The mechanism of) transcription is universal [3] (The mechanism of) translation is universal

Question 28

Suggest and explain one reason why bacteria might not be able to produce every human protein [1]

Answer 28

[1] Cannot splice (pre-mRNA), so cannot remove introns

Question 29

Explain the purpose of the marker gene [1]

Answer 29

[1] Shows that the gene has been taken up OR Allows detection of genetically modified cells/organisms