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Cell & Molecular Biology > PCR > Flashcards

PCR Flashcards

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1
Q

What does PCR stand for?

A

Polymerase chain reaction

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2
Q

Why is PCR needed if molecular cloning exists?

A
  • May not be enough DNA
  • DNA may be in with lots of other DNA molecules
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3
Q

What is the basic process of PCR?

A

To amplify a specific piece of DNA

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4
Q

Explain how PCR is specific

A

will only get amplification of your selected sequence

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5
Q

Explain how PCR is selective

A

it can amplify a specific sequence from a mixture of DNA sequences

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6
Q

Describe how there is an exponential growth when PCR is used?

A

The volume of DNA will double each cycle. As a result, if you start with 1 molecule of DNA, there will be 8 molecules after 3 cycles

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7
Q

How many is the typical number of PCR cycles that takes place?

A

approx. 30

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8
Q

What are the 3 stages of PCR?

A
  1. Denaturation
  2. Primer annealing
  3. Primer extension
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9
Q

At what temperature does denaturation occur?

A

95 degrees Celsius

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10
Q

At what temperature does primer annealing occur?

A

55-65 degrees Celsius

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11
Q

At what temperature does primer extension occur?

A

68-72 degrees Celsius

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12
Q

What occurs during the denaturation stage?

A

Double-stranded DNA dissociates into single-stranded DNA, which is facilitated by the high temperatures seen at this stage.

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13
Q

What occurs during the primer annealing stage?

A

Primers bind to the complementary sequence on ssDNA (single-stranded DNA). Primer binding is antiparallel.

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14
Q

Is primer annealing parallel or antiparallel?

A

antiparallel

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15
Q

What occurs during the primer extension phase?

A

DNA polymerase synthesises new strands of DNA from the 3’ end of the primers.

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16
Q

Why is the process of PCR considered a semi-conservative process?

A

It is made up of a new strand of DNA & another of old DNA.

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17
Q

What are the 7 things needed for PCR?

A
  • Template (DNA to copy)
  • DNA polymerase (enzyme to copy DNA)
  • Primers (Polymerase requires a free 3’ OH to start so that we know what the sequence is
  • Deoxyribonucleoside triphosphates (dNTPs)
  • Buffer
  • Thermocycler machine
  • Temperature
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18
Q

Why is it necessary to have primers for PCR?

A

Polymerase requires a free 3’ OH to start. As a result we need to know what the sequence is

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19
Q

Why is it necessary to have deoxyribonucleoside triphosphates (dNTPs) in PCR?

A

They are the DNA bases to make the new DNA strand

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20
Q

Why is it necessary to have a buffer in PCR?

A

PCR requires the correct pH & ions

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21
Q

What ion is commonly used in PCR?

A

magnesium chloride

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22
Q

Why is temperature important in PCR?

A

each PCR cycle consists of stages that each need a specific temperature

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23
Q

Why are thermostable polymerases used from thermophilic organisms during PCR?

A

otherwise there is a danger that the high temperatures experienced in PCR at certain points may denature the DNA polymerase

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24
Q

What 4 properties are important in DNA polymerases used in PCR?

A
  • thermostability
  • extension rate
  • processivity
  • fidelity
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25
Q

Why is thermostability important in DNA polymerase used in PCR?

A

There are high temperatures associated with PCR

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26
Q

What is extension rate, when discussing DNA polymerase used in PCR?

A

how fast the DNA polymerase can replicate a template

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27
Q

What is processivity, when discussing DNA polymerase used in PCR?

A

how often the DNA polymerase falls off & has to re-associate

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28
Q

What is fidelity, when discussing DNA polymerase used in PCR?

A

how accurate it is (a higher fidelity would mean that less mutations were introduced into the PCR products

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29
Q

What are the 2 main DNA polymerases?

A

Taq & Pfu

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30
Q

What are the advantages of using Taq as a DNA polymerase?

A
  • rapid extension
  • high processivity (efficiency)
  • adds an adenine overhang
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31
Q

What are the disadvantages of using Taq as a DNA polymerase?

A
  • no proof-reading activity
  • low fidelity (accuracy)
32
Q

What are the advantages of using Pfu as a DNA polymerase?

A
  • high fidelity (accuracy)
  • very thermostable
33
Q

What are the disadvantages of using Pfu as a DNA polymerase?

A
  • low processivity (efficiency)
  • products are blunt-ended
  • lower extension rate
34
Q

What are the 4 main important properties of PCR primers?

A
  • minimum size for specificity
  • specific to your template
  • come in pairs
  • appropriate melting temperature
35
Q

What is the minimum size of a PCR primer for it to provide specificity?

A

17 base pairs is an absolute minimum - usually around 20 base pairs.

36
Q

What will occur if there is only one PCR primer?

A

Only one strand will be replicated.

37
Q

What is the melting temperature (Tm)?

A

the temperature at which the primer will dissociate from the DNA template

38
Q

What is the usual range of melting temperatures of PCR primers?

A

60-64 degrees Celsius

39
Q

What temperature should the annealing temperature be (relative to melting temperature)?

A

5 degrees Celsius lower than the melting temperature

40
Q

What is the name given to the temperature at which 50% of PCR primer is annealed & 50% is not?

A

Tm = melting temperature

41
Q

What is the consequence of the melting temperature being too high?

A

self-annealing may occur

42
Q

What is the consequence of the melting temperature being too low?

A

primer often isn’t very specific

43
Q

What occurs if the annealing temperature is too low?

A

the primers may non-specifically bind to other DNA sequences

44
Q

What occurs if the annealing temperature is too high?

A

the primers may not bind efficiently (or at all) reducing product yield

45
Q

What is the old fashioned way of calculating the primer melting temperature (Tm)?

A

(2+4)
+ 4 degrees Celsius per G/C
+ 2 degrees Celsius per A/T

46
Q

What is the modern way of calculating Primer Tm?

A

use software programmes to calculate the most suitable temperature

47
Q

What is the problem with cloning using PCR?

A
  • may not be convenient restriction sites
  • not always directional
  • might not have enough DNA
  • DNA may be mixed in with lots of other DNA molecules
48
Q

How can we clone a PCR product?

A

by ligating a PCR product into a vector

49
Q

What problem may evolve as a result of trying to put a PCR product into a vector?

A

The insertion may be in the wrong orientation

50
Q

What can be done to primers, to aid in the insertion of PCR products into vectors?

A

there can be restriction enzymes inserted into the primers

51
Q

What can be done to prevent the PCR products being inserted into the vector in the wrong orientation?

A

Use 2 different primers

52
Q

What is an example of a restriction enzyme on a forward primer?

A

EcoRI

53
Q

What is an example of a restriction enzyme on a forward primer?

A

BamHI

54
Q

What are the advantages of incorporating restriction enzymes into primers?

A
  • more efficient ligation (sticky ends after restriction digest & the vector cannot ligate to itself
55
Q

What are 2 variants of PCR?

A
  • reverse PCR (PT-PCR)
  • quantitative PCR (qPCR)
56
Q

What is an overview of reverse PCR?

A
  • RNA is reverse transcribed into cDNA.
  • PCR used to amplify specific cDNA sequence
57
Q

What is cDNA?

A

complimentary DNA

58
Q

What are the potential uses of RT-PCR?

A
  • molecular cloning of protein coding cDNA sequence
  • analysis of RNA sequence/expression
59
Q

What happens to the first strand in RT-PCR?

A
  • reverse transcriptase synthesis of first strand of DNA.
  • Poly (dt) primers bind poly(A) tail of mRNA.
  • RNA is removed
60
Q

What happens to the end of the first strand during reverse transcription PCR?

A

Loops back to form the start of another strand - forming a hairpin

61
Q

What fragment synthesizes the second strand during RT-PCR?

A

Klenow fragment of DNA polymerase I

62
Q

What is special about the Klenow fragment?

A

Retains useful polymerase function, however it has lost 5’ - 3’ exonuclease activity.

63
Q

What is the ct value in quantitative PCR?

A

When the fluorescence generated by the DNA produced exceeds over the background

64
Q

What is the fluorescent dye used in qPCR?

A

SYBR Green

65
Q

When does the fluorescent dye SYBR Green fluoresce?

A

Fluoresce when bound to double-stranded DNA

66
Q

What can be attached to one end of a fluorescent probe to prevent fluorescence from occurring?

A

Quencher

67
Q

When does the quencher on a fluorescent probe get removed?

A

when the template gets replicated, the polymerase chops it off

68
Q

What does the actual letters ‘Ct’ stand for?

A

cycle threshold

69
Q

Why is qPCR still useful even though it can’t accurately measure the amount of DNA present?

A

The Ct value can be used as a relative measure to compare different DNA strands

70
Q

What is the equation of calculating the fold difference of the Ct value?

A

2 (to the power) - delta CT

71
Q

What is the most accurate way of finding out the fold difference?

A

2 (to the power) - delta delta Ct

72
Q

What equations is fold difference made up of?

A

delta delta Ct = delta Ct A - delta Ct B

73
Q

What is the equation for delta Ct A?

A

delta Ct A = Ct A test - Ct A ref

74
Q

What is the equation for delta Ct B?

A

delta Ct B = Ct B test - Ct B ref

75
Q
A

Cell & Molecular Biology (20 decks)

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