21.3 in vitro gene cloning - PCR reaction Flashcards

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1
Q

what are the 5 components needed for PCR?

A

1) DNA fragment
2) taq polymerase (DNA polymerase)
3) primers
4) nucleotides (all bases)
5) thermocycler

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2
Q

why are taq polymerase used and where are they found?

A

found in hot springs; thermostable & does not denature

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3
Q

what are primers?

A

short dna sequences with bases complementary to the end of dna fragments

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4
Q

why are nucleotides needed for PCR reactions?

A

dna synthesis

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5
Q

what is a thermocycler?

A

computer controlled machine that varies temperature

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6
Q

give 2 advantages of in vitro cloning.

A

1) extremely rapid - useful for crime scene forensics
2) no living cells needed

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7
Q

what is a disadvantage of using in vitro cloning?

A

contaminating DNA is amplified as well -> gives false results

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8
Q

what are the 3 steps of PCR?

A

1) denaturation
2) annealing
3) elongation

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9
Q

what temperature is needed in denaturation and what occurs?

A

95°C
- 2 strands of dna separates due to breaking of hydrogen bonding

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10
Q

what is the temperature needed for annealing and what occurs?

A

53°C
mixture is cooled & primers anneal to complementary bases at the end of dna fragments
- primers provide starting sequence for taq polymerase & prevent 2 strands from re-joining

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11
Q

what temperature is needed for elongation & what happens during it?

A

72°C
nucleotides are added along dna strands

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12
Q

what is the optimum temperature for taq polymerase? which PCR stage does it correspond to?

A

72°C - elongation

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