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A-level Biology > 8B - Amplifying DNA fragments (steps after isolation) > Flashcards

8B - Amplifying DNA fragments (steps after isolation) Flashcards

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1
Q

What does in vivo amplification involve?

A

Transforming host cells.

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2
Q

What do you need to do once you’ve isolated your DNA fragment?

A

Amplify it.

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3
Q

What does it mean by amplification of the DNA fragment?

A

Make lots of copies of it.

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4
Q

Why does the DNA fragment need to be amplified?

A

So you have a sufficient quantity to work with.

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5
Q

What is one method of amplification?

A

In vivo cloning

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6
Q

What is in vivo cloning?

A

Where copies of the DNA fragment are made inside a living organism.

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7
Q

Explain the process of preparing for insertion

A
  • RNA polymerase must attach to the DNA near a gene (promoter region) for transcription to take place.
  • If we want the DNA fragment to be transcribed, we need to attach it to the necessary promoter region in order to start the process.
  • Similar process when transcription is finished (terminator region).
  • Need to ensure we attach a terminator region to the other end of our DNA fragment to stop transcription.
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8
Q

What is a vector used for in insertion?

A

Used to transport DNA into a host cell.

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9
Q

What is used to transport DNA into a host cell in insertion?

A

A vector.

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10
Q

What is the most commonly used vector in insertion?

A

Plasmids.

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11
Q

What are plasmids?

A

A circular piece of DNA in bacteria.

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12
Q

Why is using plasmids as vectors in insertion useful?

A

As they nearly always contain antibiotic resistance genes.

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13
Q

Explain the process of insertion

A

1) The DNA fragment is inserted into vector DNA.
2) The vector DNA is cut open using the same restriction endonuclease that was used to isolate the DNA fragment containing the target gene. So the sticky ends of the vector are complementary to the sticky ends of the DNA fragment containing the gene.
3) The vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA (joins gene to plasmid). This process is called ligation.
4) The new combination of bases in the DNA (vector DNA + DNA fragment) is called recombinant DNA. A hybrid plasmid is formed and the gene is also successfully formed.

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14
Q

What can be used as vectors in insertion?

A

Plasmids or bacteriophages.

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15
Q

What are bacteriophages?

A

Viruses that infect bacteria.

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16
Q

What is ligation?

A

The vector DNA and DNA fragment are mixed together with DNA ligase. DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA. This process is called ligation.

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17
Q

What is DNA ligase/what does it do?

A

DNA ligase joins the sticky ends of the DNA fragment to the sticky ends of the vector DNA (causes ligation).

(Joins the sugar-phosphate backbone.)

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18
Q

What is the new combination of bases in the DNA (vector DNA + DNA fragment) called?

A

Recombinant DNA.

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19
Q

What happens if there is successful joining of a gene to the plasmid in insertion?

A

Recombinant DNA formed –> Hybrid plasmid formed –> Gene successfully formed.

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20
Q

What happens if there is unsuccessful joining of a gene to the plasmid in insertion?

A

Unsuccessful joining –> Original plasmid reformed –> Circularised DNA also formed.

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21
Q

What happens to the plasmid when the restriction enzymes cut open the plasmid in insertion?

A

One of the antibiotic resistance genes is disrupted.

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22
Q

What do the antibiotic resistance genes that aren’t disrupted when the plasmid is cut open in insertion do?

A

They are used in selection of the correct host cells.

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23
Q

Explain the process of transformation

A

1) The vector with the recombinant DNA is used to transfer the gene into host cells.
2) If a plasmid vector is used, host cells have to be persuaded to take in the plasmid vector and its DNA (plasmids and bacterial cells are mixed together in an ice-cold medium containing calcium ions - calcium chloride solution. Calcium ions, combined with an increase in temperature (heat-shocked to around 42 degrees C for 1-2mins), makes the bacterial membrane permeable and therefore allows the plasmids to pass through into the cytoplasm.)
3) With a bacteriophage vector, the bacteriophage will infect the host bacterium by injecting its DNA into it. The phage DNA (with the target gene in it) then integrates into the bacterial DNA.
4) Host cells that take up the vectors containing the gene of interest are said to be transformed.

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24
Q

Will all the bacterial cells have the desired gene at the end of transformation?

A

No

25
Q

Why won’t all the bacterial cells have the desired gene at the end of transformation?

A
  • Only a few bacterial cells (as little as 1%) take up the plasmids when the two are mixed together.
  • Some plasmids will have closed up again without incorporating the DNA fragment.
  • Sometimes the DNA fragment ends can join together to form its own plasmid.
26
Q

Around how many host cells will take up the vector and its DNA in transformation?

A

Around 5%.

27
Q

Why is it important to be able to identify which cells have been transformed?

A

Because only around 5% of host cells will take up the vector and its DNA in transformation.

28
Q

What can be used to identify transformed genes in identification?

A

Marker genes - antibiotic-resistance markers, fluorescent markers and enzyme markers.

29
Q

What is the process of using DNA technology?

A

1) Isolation
2) Insertion
3) Transformation
4) Identification
5) Growth/cloning

30
Q

How can marker genes be used to identify transformed host cells?

A

1) They can be inserted into vectors at the same time as the gene to be cloned. This means any transformed host cells will contain the gene to be cloned and the marker gene.
2) Host cells are grown on agar plates. Each cell divides and replicates its DNA, creating a colony of cloned cells. Transformed cells will produce colonies where all the cells contain the cloned gene and the marker gene.
3) The marker gene can code for antibiotic resistance - host cells are grown on agar plates containing the specific antibiotic, so only transformed cells that have the marker gene will survive and grow. Or it can code for fluorescence - when the agar plate is placed under UV light only transformed cells will fluoresce.

4) Identified transformed cells are allowed to grow more, producing lots and lots of copies of the cloned gene.

31
Q

What are the 3 gene markers used in identification?

A

Antibiotic resistance markers
Fluorescent markers
Enzyme markers

32
Q

Explain how antibiotic resistance markers can be used to identify which plasmids have taken up the DNA fragment

A
  • The second antibiotic resistance gene is used to identify plasmids with a DNA fragment.
  • If the DNA fragment has been taken inserted into the resistance gene it will no longer grow on a medium containing the antibiotic.
  • If we do grow it on this medium, we will kill the cells that are not resistant (has the DNA fragment in plasmid).
  • In order to identify the bacteria with our desired gene we use replica plating.
33
Q

Explain how fluorescent markers can be used to identify which plasmids have taken up the DNA fragment

A
  • Gene in jellyfish which produces green fluorescent protein (GFP) has been incorporated into a plasmid.
  • Gene to be cloned is transplanted into the centre of the GFP gene, disrupting its transcription.
  • If hybrid plasmid is taken up, the bacteria will not glow and can be identified.
  • If plasmid has not been taken up, the bacteria will glow and would not be used.
  • Results can be viewed under a microscope, making process quicker.
34
Q

Explain how enzyme markers can be used to identify which plasmids have taken up the DNA fragment

A
  • Enzyme lactase turns a colourless substance blue.
  • Required gene transplanted into gene that makes lactase, disrupting it.
  • If the plasmid has been taken up, lactase will not be produced so the substrate will remain colourless.
35
Q

How is the growth/cloning stage carried out?

A

Using PCR.

36
Q

What do you need if you want the transformed host cells to produce the protein coded for by the DNA fragment?

A

The vector needs to contain specific promoter and terminator regions.

37
Q

What are promoter regions?

A

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

38
Q

What are terminator regions?

A

DNA sequences that tell the enzyme RNA polymerase when to stop producing mRNA.

39
Q

What won’t happen without the right promoter region?

A

The DNA fragment won’t be transcribed by the host cell and a protein won’t be made.

40
Q

Where might promoter and terminator regions be present?

A

In the vector DNA.

41
Q

What happens if promoter and terminator regions aren’t present in the vector DNA?

A

They may have to be added in along with the fragment.

42
Q

In vivo gene cloning

A

Transferring to a host cell using a vector, uses a cell.

43
Q

In vitro gene cloning

A

Using PCR, outside of cell.

44
Q

What is the polymerase chain reaction?

A

Method of copying DNA - automated process, making it more rapid and efficient than using a vector.

45
Q

What does in vitro amplification use?

A

PCR

46
Q

What does PCR stand for?

A

Polymerase chain reaction.

47
Q

What do we need for PCR?

A
  • DNA fragment to be copied.
  • DNA polymerase to join nucleotides together (for PCR we use taq polymerase, taken from bacteria that live in hot springs so its tolerant to heat - thermostable).
  • Primers (short pieces of DNA that bind to the start of each end of the fragment and also help to prevent both ends from joining back up).
  • Nucleotides (all 4 bases found in DNA).
  • Thermocycler (computer-controlled machine that varies temperatures precisely over a period of time).
48
Q

What is the type of DNA polymerase used in PCR?

A

For PCR we use taq polymerase, taken from bacteria that live in hot springs so its tolerant to heat - thermostable

49
Q

What are primers?

A

Primers (short pieces of DNA that bind to the start of each end of the fragment and also help to prevent both ends from joining back up).

50
Q

What is a thermocycler?

A

Thermocycler (computer-controlled machine that varies temperatures precisely over a period of time).

51
Q

How many steps are there to PCR?

A

3

52
Q

What are the 3 steps of PCR?

A

1) Separation of the strands
2) Annealing of the primers
3) Synthesis

53
Q

Explain the 1st stage of PCR - separation of the strands

A
  • DNA fragment, primers and taq polymerase placed into vessel in thermocycler.
  • Temperature raised to 95 degrees C.
  • This breaks the hydrogen bonds between the strands causing the 2 DNA strands to separate.
54
Q

Explain the 2nd stage of PCR - annealing of the primers

A
  • 2 primers are added to the 2 separated strands (one on each strand at the 5’ end).
  • Temperature cooled to 55 degrees C which helps the primers anneal to their complementary bases on the DNA fragment.
  • The primers provide a starting sequence to tell taq polymerase where to start copying the DNA.
  • Primers prevent the 2 strands from rejoining as its now double stranded in those places.
55
Q

Explain the 3rd stage of PCR - synthesis

A
  • Temperature raised to 72 degrees C, the optimum temperature for taq polymerase.
  • 2 polymerase molecules attach to the 2 primers on the 2 DNA strands and move along the strand.
  • As they move along they create new ‘complementary’ DNA using the free nucleotides added.
56
Q

Explain the use of PCR in in vitro cloning

A
  • DNA fragment, primers and taq polymerase placed into vessel in thermocycler.
  • Temperature raised to 95 degrees C.
  • This breaks the hydrogen bonds between the strands causing the 2 DNA strands to separate.
  • 2 primers are added to the 2 separated strands (one on each strand at the 5’ end).
  • Temperature cooled to 55 degrees C which helps the primers anneal to their complementary bases on the DNA fragment.
  • The primers provide a starting sequence to tell taq polymerase where to start copying the DNA.
  • Primers prevent the 2 strands from rejoining as its now double stranded in those places.
  • Temperature raised to 72 degrees C, the optimum temperature for taq polymerase.
  • 2 polymerase molecules attach to the 2 primers on the 2 DNA strands and move along the strand.
  • As they move along they create new ‘complementary’ DNA using the free nucleotides added.
57
Q

How many new copies of the fragment of DNA are formed from one cycle of PCR?

A

2

58
Q

What happens once 1 cycle of PCR is complete?

A

The cycle starts again, with the mixture being heated to 95 degrees C and this time all four strands (2 original and 3 nes) are used as templates.

59
Q

How does the amount of DNA increase with each PCR cycle?

A

Each PCR doubles the amount of DNA.

A-level Biology (42 decks)

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