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A-level Biology Oxford AQA > 3.4.10 Recombinant DNA Technology > Flashcards

3.4.10 Recombinant DNA Technology Flashcards

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1
Q

What is recombinant DNA?

A

DNA from 2 different sources are combined

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2
Q

What does the production of DNA fragments involve?

A

Transferring a fragment of DNA from one organism to another

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3
Q

Explain why the recipient and donor organisms don’t have to be same species when transferring a fragment of DNA from one organism to another

A
  • ∵ genetic code is universal
    • (same DNA base triplets code for same amino acids in all living things)
  • ∵ transcription and translation mechanisms are pretty similar
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4
Q

Name 3 methods of making DNA fragments

A
  • Using Reverse Transcriptase
  • Using Restriction Endonuclease Enzymes
  • Using a ‘Gene Machine’
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5
Q

Why is mRNA easier to obtain than DNA?

A

Many mRNA molecules (complementary to a gene)

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6
Q

What does reverse transcriptase do?

A

Makes DNA from an RNA template

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7
Q

What is the DNA produced by reverse transcriptase called?

A

complementary DNA (cDNA)

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8
Q

Describe how you can make DNA fragments using reverse transcriptase

A
  1. mRNA is isolated from cells
  2. Its mixed with free DNA nucleotides and reverse transcriptase
  3. Reverse transcriptase uses mRNA as a template to synthesis a new strand of cDNA
  4. DNA polymerase is used to build up the complementary base pairings with cDNA = DNA is formed
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9
Q

Some sections of DNA have ________ sequences of nucleotides

A

palindromic

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10
Q

What are palindromic sequences of nucleotides?

A

Sequences that consist of antiparallel base pairs

(Base pairs that read the same in opposite directions)

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11
Q

What are restriction endonucleases?

A

Enzymes that recognise specific palindromic sequences (known as recognition sequences) and cut (digest) DNA at these places

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12
Q

Why do different restriction endonucleases cut at different specific recognition sequences?

A

∵ shape of recognition sequence is complementary to enzyme’s active site

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13
Q

When can you use restriction endonucleases to create a DNA fragment?

A

If recognition sequences are present either side of DNA fragment

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14
Q

Describe how you can use restriction endonuclease to produce a DNA fragment

A
  1. DNA sample is incubated with specific restriction endonuclease & it cuts DNA fragment out via hydrolysis reaction
  2. Sometimes cut leaves sticky ends - small tails of unpaired bases at each end of the fragment
  3. Sticky ends can be used to bind (anneal) DNA fragment to another pieces of DNA that has sticky ends with complementary sequences
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15
Q

What is meant by a ‘gene machine’?

A
  • Technology that enables fragments of DNA that can be synthesised from scratch without need for pre-existing DNA template
    • Database contains necessary info to produce DNA fragments
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16
Q

What does a ‘gene machine’ allow you to do?

A

Can produce DNA sequences that don’t exist naturally

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17
Q

Describe how you can use a ‘gene machine’ to produce a DNA fragment

A
  1. Sequence required is designed
  2. 1st nucleotide in sequence is fixed to some sort of support
    • e.g. a bead
  3. Nucleotides are added step by step in correct order, in a cycle of processes that include adding protecting groups
  4. Short sections of DNA (called oligonucleotides - 20 nucleotides long) are produced
  5. Once complete, they’re broken off from support and all protecting groups are removed
  6. Oligonucleotides can then be joined together to make longer DNA fragments
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18
Q

Using a ‘Gene Machine’ to Producing DNA Fragments

Explain why protecting groups are added when nucleotides are added step by step in correct order

A

Protecting groups make sure nucleotides are joined at the right points, to prevent unwanted branching

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19
Q

Name 2 methods of amplifying (makes lots of copies of) DNA fragments

A
  • In Vivo Amplification
  • In Vitro Amplification
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20
Q

Name the 3 stages in in vivo amplification

A
  1. DNA Fragment is Inserted into Vector
  2. Vector Transfers DNA Fragment into Host Cells
  3. Identifying Transformed Host Cells
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21
Q

In Vivo Amplification

What vectors are normally used?

A

Can be plasmids or bacteriophages (viruses that infect bacteria)

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22
Q

What is a recombinant plasmid?

A

When a gene is added from another organism to the plasmid

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23
Q

In Vivo Amplification

1) Describe how a DNA fragment is inserted into a vector

A
  1. Vector DNA is cut open using same restriction endonuclease that was used to isolate DNA fragments containing the target gene
    • So sticky ends of vector are complementary to sticky ends of DNA fragment containing the gene
  2. Vector DNA and DNA fragment are mixed together with DNA ligase
    • DNA ligase joins sticky ends of DNA fragment to sticky ends of vector DNA
      • aka ligation
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24
Q

Describe how a plasmid vector transfers recombinant DNA into host cells

A

Host cells are to be persuaded to take in the plasmid vector and its DNA

25
Q

Describe how a bacteriophage vector transfers recombinant DNA into host cells

A
  • Bacteriophage infect host bacterium by injecting its DNA into it
  • Phage DNA (with the target gene in it) then integrates into bacterial DNA
26
Q

Host cells that take up vectors containing the gene of interest are said to be ______

A

transformed

27
Q

What are used to identify transformed cells?

A

Marker genes

28
Q

When are marker genes inserted into vectors and why?

A
  • Inserted at same time as target gene (i.e. gene to be cloned)
  • ∴ transformed host cells will contain target gene and marker gene
29
Q

What are host cells grown on?

A

Agar plates

Each cell divides and replicates its DNA, creating colony of cloned cells

30
Q

Name 3 examples of marker genes

A
  • Genes that code for antibiotic resistance
  • Genes that code for fluorescence
  • Enzyme markers
31
Q

Describe how genes that can code for antibiotic resistance can be used as marker genes

A

Host cells are grown on agar plates containing specific antibiotic so only transformed cells with marker genes will grow

32
Q

Describe how genes that can code for fluorescence can be used as marker genes

A

Under UV light only transformed cells will fluoresce

33
Q

Describe how enzyme markers can be used as marker genes

A

Gene produces lactase and lactase will turn colourless substate blue

34
Q

Describe replica plating

A
  1. Bacteria are placed into a medium containing e.g. ampicillin, with all bacteria that have taken up the plasmid (with gene for resistance to ampicillin) surviving
  2. Each separate cell then grows into a genetically identical colony
  3. A tiny sample of each colony is then placed into a medium containing tetracycline
  4. The cells that are destroyed contain the recombinant DNA
    • Shows new gene has been taken up as gene for resistance to tetracycline is made useless
35
Q

What does a vector need to contain, if you want transformed host cells to produce a protein coded by DNA fragment?

A

Vector contains specific promoter and terminator regions

36
Q

Producing Proteins requires Promotor and Terminator Regions

What happens if the vector doesn’t contain the right promoter region?

A

DNA fragment won’t be transcribed by host cell and protein won’t be made

37
Q

What do promoter regions do?

A

DNA sequences that tell enzyme RNA polymerase when to start producing mRNA

(Terminator regions tell it when to stop)

38
Q

Producing Proteins requires Promotor and Terminator Regions

Promoter and terminator regions may be present in the ____ ____ or may be added in along with the _____

A

Promoter and terminator regions may be present in the vector DNA or may be added in along with the fragment

39
Q

How are copies of DNA fragments in in vitro made?

A

Use polymerase chain reaction (PCR)

(copies of DNA fragments are made outside of living organisms)

40
Q

What is the benefit of using PCR?

A

It can be used to make millions of copies of a fragment of DNA in a few hours

41
Q

Describe how DNA fragments are made in in vitro amplification

A
  1. Reaction mixture set up containing DNA sample, free nucleotides, primers and DNA polymerase
    • DNA polymerase creates new DNA strands
  2. DNA mixture is heated to 95°C to break hydrogen bonds between 2 strands of DNA
    • Strands separate
  3. Mixture is cooled to between 50°C and 65°C so primer can bind (anneal) to strands
  4. Nucleotides attach by complementary base pairing
  5. Reaction mixture is heated to 72°C so DNA polymerase can work
  6. DNA polymerase join free DNA nucleotides alongside each template strand
  7. 2 new copies of fragment of DNA formed & 1 cycle of PCR is complete
  8. Cycle starts again, with mixture heated to 95°C and all 4 strands (2 original and 2 new) are used as templates
  9. Each PCR cycle doubles the amount of DNA
42
Q

In Vitro Amplification

In the 3rd PCR cycle, how many DNA fragments are produced?

A

16 DNA fragments

  • 1st cycle = 2 x 2 = 4 DNA fragments
  • 2nd cycle = 4 x 2 = 8 DNA fragments
  • 3rd cycle = 8 x 2 = 16 DNA fragments
43
Q

How are transformed organisms produced?

A

Through genetic engineering

44
Q

Describe how transformed microorganisms can be produced

A
  • Can be made using same technology as in vivo cloning
  • e.g. foreign DNA inserted into microorganisms to produce lots of useful protein
45
Q

Describe how transformed plants can be produced

A
  • Gene codes for desirable protein inserted into plasmid
  • Plasmid is added to bacterium and bacterium is used as vector to get gene into plant cells
  • If right promoter region has been added along with gene, transformed cells will be able to produce the desired protein
46
Q

Describe how transformed animals can be produced

A
  • Gene codes for desirable protein can be inserted into early animal embryo or into egg cells of a female
  • If gene inserted into a very early embryo, all body cells of resulting transformed animal will contain the gene
  • Inserting into egg cells = when female reproduces = all cells of offspring contain the gene
47
Q

Promoter regions that are only activated in specific cell types can be used to control…

A

exactly which of animal’s body cells the protein is produced in

48
Q

What is the benefit of producing a protein in certain cells?

A

It can be harvested more easily

49
Q

What happens when a protein is produced in the wrong cells?

A

Damages the organism

50
Q

State some agricultural benefits of using recombinant DNA technology

A
  1. Agriculture crops can be transformed to they give higher yields or are more nutritious
    • Means plants can be used to reduce risk of famine and malnutrition
  2. Crops can be transformed to have pest resistance = fewer pesticides are needed
    • Reduces costs and reduces any environmental problems associated with using pesticides
51
Q

State some industrial benefits of using recombinant DNA technology

A
  • Industrial processes often use enzymes
  • Enzymes can be produced from transformed organisms = produced in large quantities for less money = reducing costs
52
Q

State some medical benefits of using recombinant DNA technology

A
  1. Many drugs and vaccines are produced by transformed organisms, using recombinant DNA technology
    • Can made quickly, cheaply and in large quantities using this method
    • So more people can afford them
  2. Recombinant DNA technology has the potential to be used in gene therapy to treat human diseases
53
Q

What does gene therapy involve?

A

Altering the defective genes (mutated alleles) inside cells to treat genetic disorders and cancer

54
Q

State some agricultural disadvantages of using recombinant DNA technology

A
  1. Farmers might plant only 1 type of transformed crop (monoculture)
    1. Makes whole crop vulnerable to same disease ∵ plants are genetically identical
    2. Monoculture also reduces biodiversity = damages the environment
55
Q

State some industrial disadvantages of using recombinant DNA technology

A
  1. Anti-globalisation activists oppose globalisation
    1. A few, large biotechnology companies control some forms of genetic engineering
    2. As use of this technology increases = companies get bigger and more powerful
    3. May force smaller companies out of business
  2. Without proper labelling, some people think they won’t have a choice about whether to consume food made using genetically engineered organisms
  3. Some consumer markets (e.g EU) won’t import GM food and products
    1. Can cause economic loss to producers who have traditionally sold to those markets
56
Q

State some medical disadvantages of using recombinant DNA technology

A
  1. Companies who own genetic engineering technologies may limit the use of technologies that could be saving lives
  2. Some people worry this technology could be use unethically
    1. e.g. make designer babies = illegal
57
Q

What is the role of a primer in PCR?

A

Enables replication/sequencing to start

58
Q

What is are DNA primers?

A

Short lengths of single-stranded DNA that are complementary to a particular sequence of DNA

A-level Biology Oxford AQA (41 decks)

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