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TB1 > TB1-2: PCR > Flashcards

TB1-2: PCR Flashcards

1
Q

What does PCR stand for?

A

Polymerase Chain Reaction

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2
Q

What is the polymerase chain reaction (what is it used for and by what method)?

A

Amplifying the number of short, specific DNA sequences by using a DNA replication-dependent method

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3
Q

Name one broad area of research where PCR is heavily used

A

Molecular Biology

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4
Q

PCR continues to have increased importance in disciplines other than Molecular Biology, give an example of a different field where it is used

A

Medicine

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5
Q

What characteristic of PCR is both an advantage and disadvantage?

A

Sensitivity

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6
Q

How long does PCR generally take?

A

2-3 hours or as short as 20 mins with new machines

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7
Q

What feature of PCR makes it “safe”?

A

non-radioactive

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8
Q

What makes PCR “specific”?

A

it only yields products of the target DNA due to specific primers

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9
Q

What evidence is there that PCR is “efficient”?

A

one double strand DNA template can be used to produce a billion copies of DNA in under 2 hours

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10
Q

Define “efficiency”

A

A measure of performance and effectiveness of a system or component.
The ratio of useful output given the amount (per) required input

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11
Q

Using the definition of efficiency, explain why PCR is efficient

A

Efficiency is calculated as the ratio of useful output per required input. In the case of PCR, there is a very small amount of require input to give a large useful output - one double strand of DNA can be used as a template to form billions of copies in under 2 hours.

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12
Q

What is advantageous about PCR being sensitive?

A

extremely small amounts of starting material (DNA) can be used to produce large amounts of product

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13
Q

What is disadvantageous about PCR being sensitive?

A

Issues with contamination
“PCR product carryover contamination”
If small amounts of contaminant are present in the starting material, this will also be amplified, leading to accumulation of contaminated product

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14
Q

Define “sensitivity” in relation to identifying patients with a disease.

A

The ability to correctly identify patients with a disease
True Positive

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15
Q

Define “specificity” in relation to identifying patients wih a disease

A

The ability to correctly identify patients without the disease
True Negative

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16
Q

What are the 3 key phases in the PCR reaction? (in order)

A

Denaturation
Annealing
Elongation
Repeat

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17
Q

What temperatures does the stage of denaturation take place in during PCR?

A

95 Celsius

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18
Q

What temperature does the stage of annealing take place in during PCR?

A

50-65 Celsius

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19
Q

What temperature does the stage of elongation take place in during PCR?

A

72 Celsius

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20
Q

What process does the Polymerase Chain Reaction rely on?

A

DNA replication

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21
Q

PCR relies on DNA replication, therefore, what 4 basic starting materials are required to carry out the reaction?

A

Oligonucleotide Primers to provide the 3’OH
Template DNA
DNA polymerase to copy the template DNA
Deoxyribonucleotides

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22
Q

What other name is given to the process of denaturation in PCR?

A

“melting” or “strand separation”

23
Q

How long does denaturation in PCR take place for?

A

30 sec

24
Q

What is being ‘annealed’ to DNA during the annealing stage of PCR?

A

(oligonucleotide) primers

25
Q

How long does the stage of annealing last for during PCR?

A

30 sec

26
Q

What other name can be given to the “elongation” stage of PCR?

A

primer extension

27
Q

How long does elongation take during PCR?

A

1 min/kb
N.B. it is different for each enzyme

28
Q

Why are tough enzymes (polymerases) required for PCR?

A

to deal with the high temperatures

29
Q

Tough enzymes are needed in PCR to deal with the high temperatures, where are polymerases isolated from (species)?Example?

A

Thermophilic bacteria
e.g. Taq from Thermus aquaticus
Pfu from Pyrococcus furiosus

30
Q

What is the ‘template DNA’ in a PCR reaction?

A

the sample to be amplified

31
Q

What is the ‘target DNA’ in a PCR reaction?

A

the region that you want to amplify on the template DNA
NB this could be a shorter sequence within the template

32
Q

Name two thermostable DNA polymerases

A

Taq Thermus aquaticus
Pfu Pyrococcus furiosus
Pwo Pyrococcus woesei
Phusion (human-made)
Expad HiFi, Expand 20kbplus

33
Q

What aspect of using Taq as a DNA pol in PCR makes it error prone?
How might this be used to advantage rather than a disadvantage?

A

it has no proofreading
can be used to generate random mutations

34
Q

What is ‘subcloning’?

A

A procedure in molecular biology where DNA is transferred from one vector to another in order to gain the functionality required to study the sequence of interest

35
Q

What aspect of Taq DNA pol makes it useful for some subcloning reactions such as GEM-T?

A

it leaves a 3’ A overhang

36
Q

What major advantage does using Pfu and Pwo over Taq DNA pol in PCR have?

A

Pfu and Pwo have proof-reading and are therefore accurate which is important in order to generate faithful copies of template

37
Q

Why does using Pfu or Pwo instead of Taq in PCR lead to lower efficiency?

A

They leave no overhangs, therefore blunt end cloning is required which has lower efficiency than 3’A overhang

38
Q

In PCR primer design, what is the primer sequence complementary to?

A

the DNA adjacent to the region going to be amplified

39
Q

Why must a PCR primer be ‘sufficiently long’? ANd what is the usual range for this length?

A

Long so that it only binds to the single site within the template DNA
18-30 nucleotides

40
Q

What percentage must G and C content be for PCR primer design?

A

between 40%-60%

41
Q

Which base must be present at the 3’end of primers for PCR?

A

G or C

42
Q

Why must G or C be at the 3’ end on primers used for PCR?

A

GC clamp stabilises primer binding

43
Q

What are inverted repeats?

A

A single stranded sequence of nucleotides which is followed by its reverse complement downstream (e.g. TTACGnnnnCGAA )

44
Q

During Primer design for PCR, there should be no sequence that results in secondary structures. What type of sequence would result in a secondary strucutre?

A

Inverted Repeats (causes hairpin loops)

45
Q

Define primer Tm

A

Primer melting temperature is the temperature at which half of the double stranded DNA will dissociate to become single stranded

46
Q

How do you calculate primer Tm?

A

2(A+T) + 4(G+C)

47
Q

What range of temperatures is common for primer Tm?

A

52-58 celsius
or 55-70 celsius

48
Q

How do you calculate a rough value for annealing temperature (in PCR) using primer Tm?

A

usually Tm minus 5 Celsius

49
Q

How do primers provide specificity? consider their structure

A

They are short, single stranded DNA sequences that are designed to be complementary to the start of the target region of DNA being synthesised in PCR
THey provide a starting point for the synthesis

50
Q

What buffer is used in PCR?

A

KCl, MgCl2

51
Q

Why is a buffer needed in PCR?

A

They provide a suitable chemical environment for activity of DNA polymerase

52
Q

WHat is the typical range for PCR buffer pH?

A

8.0-9.5

53
Q

Why is K+ (potassium ions) from KCl a common component in PCR buffer when Taq DNA polymerase is used?

A

it promotes primer annealing

54
Q
A

TB1 (7 decks)

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