TB1-2: PCR

Question 1

What does PCR stand for?

Answer 1

Polymerase Chain Reaction

Question 2

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

Answer 2

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

Question 3

Name one broad area of research where PCR is heavily used

Answer 3

Molecular Biology

Question 4

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

Answer 4

Medicine

Question 5

What characteristic of PCR is both an advantage and disadvantage?

Answer 5

Sensitivity

Question 6

How long does PCR generally take?

Answer 6

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

Question 7

What feature of PCR makes it “safe”?

Answer 7

non-radioactive

Question 8

What makes PCR “specific”?

Answer 8

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

Question 9

What evidence is there that PCR is “efficient”?

Answer 9

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

Question 10

Define “efficiency”

Answer 10

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

Question 11

Using the definition of efficiency, explain why PCR is efficient

Answer 11

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.

Question 12

What is advantageous about PCR being sensitive?

Answer 12

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

Question 13

What is disadvantageous about PCR being sensitive?

Answer 13

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

Question 14

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

Answer 14

The ability to correctly identify patients with a disease
True Positive

Question 15

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

Answer 15

The ability to correctly identify patients without the disease
True Negative

Question 16

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

Answer 16

Denaturation
Annealing
Elongation
Repeat

Question 17

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

Answer 17

95 Celsius

Question 18

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

Answer 18

50-65 Celsius

Question 19

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

Answer 19

72 Celsius

Question 20

What process does the Polymerase Chain Reaction rely on?

Answer 20

DNA replication

Question 21

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

Answer 21

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

Question 22

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

Answer 22

“melting” or “strand separation”

Question 23

How long does denaturation in PCR take place for?

Answer 23

30 sec

Question 24

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

Answer 24

(oligonucleotide) primers

Question 25

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

Answer 25

30 sec

Question 26

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

Answer 26

primer extension

Question 27

How long does elongation take during PCR?

Answer 27

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

Question 28

Why are tough enzymes (polymerases) required for PCR?

Answer 28

to deal with the high temperatures

Question 29

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

Answer 29

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

Question 30

What is the 'template DNA' in a PCR reaction?

Answer 30

the sample to be amplified

Question 31

What is the 'target DNA' in a PCR reaction?

Answer 31

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

Question 32

Name two thermostable DNA polymerases

Answer 32

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

Question 33

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?

Answer 33

it has no proofreading can be used to generate random mutations

Question 34

What is 'subcloning'?

Answer 34

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

Question 35

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

Answer 35

it leaves a 3' A overhang

Question 36

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

Answer 36

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

Question 37

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

Answer 37

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

Question 38

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

Answer 38

the DNA adjacent to the region going to be amplified

Question 39

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

Answer 39

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

Question 40

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

Answer 40

between 40%-60%

Question 41

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

Answer 41

G or C

Question 42

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

Answer 42

GC clamp stabilises primer binding

Question 43

What are inverted repeats?

Answer 43

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

Question 44

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?

Answer 44

Inverted Repeats (causes hairpin loops)

Question 45

Define primer Tm

Answer 45

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

Question 46

How do you calculate primer Tm?

Answer 46

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

Question 47

What range of temperatures is common for primer Tm?

Answer 47

52-58 celsius or 55-70 celsius

Question 48

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

Answer 48

usually Tm minus 5 Celsius

Question 49

How do primers provide specificity? consider their structure

Answer 49

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

Question 50

What buffer is used in PCR?

Answer 50

KCl, MgCl2

Question 51

Why is a buffer needed in PCR?

Answer 51

They provide a suitable chemical environment for activity of DNA polymerase

Question 52

WHat is the typical range for PCR buffer pH?

Answer 52

8.0-9.5

Question 53

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

Answer 53

it promotes primer annealing