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Y13 A2 Biology > Amplifying DNA fragments > Flashcards

Amplifying DNA fragments Flashcards

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1
Q

What are the two ways to amplify a DNA fragment?

A

In vivo cloning

In vitro cloning

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2
Q

What is in vivo cloning?

A

Gene copies are made inside of a living organism

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3
Q

What is in vitro cloning?

A

Gene copies are made outside of a living organism

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4
Q

What are the three steps in in vivo cloning?

A

1) Make recombinant DNA
2) Transforming cells
3) Identifying transformed cells

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5
Q

In the first step of in vivo cloning, what does the vector’s DNA need to be? (vivo)

A

Isolated

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6
Q

What are 2 examples of a vector? (vivo)

A

Bacteriophage

Plasmid

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7
Q

What is a vector? (vivo)

A

A molecule which transfers genetic material from one organism to another

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8
Q

Why is the same restriction endonuclease enzyme used as was when the fragment was created? (vivo)

A

To produce sticky ends in the vector’s DNA which are complimentary to the sticky ends of the DNA fragment which contains the target allele

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9
Q

What are the DNA fragments and vector’s DNA mixed with? (vivo)

A

DNA ligase

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10
Q

What is the role of DNA ligase? (vivo)

A

Ligation

To anneal the sticky ends of the DNA fragment with the vector

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11
Q

What name is given to the DNA fragment and the vector together? (vivo)

A

Recombinant DNA

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12
Q

When is a host cell considered transformed? (vivo)

A

When they have taken up the vector containing the target gene

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13
Q

What two conditions are needed for the bacteria to uptake the vector and why? (vivo)

A

Ice cold CaCl2 - Makes the membranes more permeable

Heat shock to 42 degrees - to encourage uptake

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14
Q

What is a host cell? (vivo)

A

The cell which the vector containing the recombinant DNA transfers the target gene into

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15
Q

What is a marker gene? (vivo)

A

It tells you whether a cell has been transformed

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16
Q

Where and when is a marker gene added? (vivo)

A

Into the vector at the same time as the target gene is added

17
Q

What are three examples of marker genes? (vivo)

A

Antibiotic resistance
Fluorescence
Radioactivity

18
Q

How can transformed cells be identified? (vivo)

A

Fluorescence - They will glow under UV light
Antibiotic resistance - They will survive in the presence of antibiotics
Radioactive - Show in an X-ray/on film

19
Q

Why is it important to be able to identify transformed cells? (vivo)

A

So transformed cells can go on to divide and produce more copies of the target gene

20
Q

What is another name for in vitro cloning?

A

Polymerase chain reaction

21
Q

Why is PCR good? (vitro)

A

It makes millions of copies of DNA outside the body quickly

22
Q

In the first step of PCR, a mixture is created. What is in this mixture? (Vitro)

A

Double stranded DNA sample
Primers
DNA polymerase
Free DNA nucleotides

23
Q

What temperature is the mixture heated to first and why? (vitro)

A

92 degrees

To break the hydrogen bonds between the double stranded DNA to produce two single strands

24
Q

What type of DNA polymerase is used and why? (vitro)

A

Thermostable

It does not denature at 92 degrees as it is collected from hot springs

25
Q

What is the second temperature the mixture is heated to and why? (vitro)

A

Between 50 and 65

To allow the primers to anneal to the single strands

26
Q

What are primers? (vitro)

A

Single stranded molecule with complimentary nucleotides to the DNA template strand

27
Q

Why are primers required? (vitro)

A
  • To show the enzyme where to start adding nucleotides

- So the strand of DNA is double stranded so the DNA polymerase has a starting strand

28
Q

What is the third temperature that the mixture is heated to and why?

A

72 degrees

Optimum for the DNA polymerase to work

29
Q

What is the role of DNA polymerase? (vitro)

A

Add free complimentary DNA nucleotides to the template strands

30
Q

How many new copies of the DNA fragments are made every PCR cycle? (vitro)

A

2

31
Q

In in vivo cloning, what do you need to make sure is added and why? (vivo)

A

Promoter and terminator regions

To produce the protein which is being coded for

32
Q

What happens if there are no promoter regions? (vivo)

A

The DNA fragment wont be transcribed by the host cell and the protein wont be made

Y13 A2 Biology (45 decks)

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