TB1-2: PCR Flashcards
What does PCR stand for?
Polymerase Chain Reaction
What is the polymerase chain reaction (what is it used for and by what method)?
Amplifying the number of short, specific DNA sequences by using a DNA replication-dependent method
Name one broad area of research where PCR is heavily used
Molecular Biology
PCR continues to have increased importance in disciplines other than Molecular Biology, give an example of a different field where it is used
Medicine
What characteristic of PCR is both an advantage and disadvantage?
Sensitivity
How long does PCR generally take?
2-3 hours or as short as 20 mins with new machines
What feature of PCR makes it “safe”?
non-radioactive
What makes PCR “specific”?
it only yields products of the target DNA due to specific primers
What evidence is there that PCR is “efficient”?
one double strand DNA template can be used to produce a billion copies of DNA in under 2 hours
Define “efficiency”
A measure of performance and effectiveness of a system or component.
The ratio of useful output given the amount (per) required input
Using the definition of efficiency, explain why PCR is efficient
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.
What is advantageous about PCR being sensitive?
extremely small amounts of starting material (DNA) can be used to produce large amounts of product
What is disadvantageous about PCR being sensitive?
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
Define “sensitivity” in relation to identifying patients with a disease.
The ability to correctly identify patients with a disease
True Positive
Define “specificity” in relation to identifying patients wih a disease
The ability to correctly identify patients without the disease
True Negative
What are the 3 key phases in the PCR reaction? (in order)
Denaturation
Annealing
Elongation
Repeat
What temperatures does the stage of denaturation take place in during PCR?
95 Celsius
What temperature does the stage of annealing take place in during PCR?
50-65 Celsius
What temperature does the stage of elongation take place in during PCR?
72 Celsius
What process does the Polymerase Chain Reaction rely on?
DNA replication
PCR relies on DNA replication, therefore, what 4 basic starting materials are required to carry out the reaction?
Oligonucleotide Primers to provide the 3’OH
Template DNA
DNA polymerase to copy the template DNA
Deoxyribonucleotides
What other name is given to the process of denaturation in PCR?
“melting” or “strand separation”
How long does denaturation in PCR take place for?
30 sec
What is being ‘annealed’ to DNA during the annealing stage of PCR?
(oligonucleotide) primers
How long does the stage of annealing last for during PCR?
30 sec
What other name can be given to the “elongation” stage of PCR?
primer extension
How long does elongation take during PCR?
1 min/kb
N.B. it is different for each enzyme
Why are tough enzymes (polymerases) required for PCR?
to deal with the high temperatures
Tough enzymes are needed in PCR to deal with the high temperatures, where are polymerases isolated from (species)?Example?
Thermophilic bacteria
e.g. Taq from Thermus aquaticus
Pfu from Pyrococcus furiosus
What is the ‘template DNA’ in a PCR reaction?
the sample to be amplified
What is the ‘target DNA’ in a PCR reaction?
the region that you want to amplify on the template DNA
NB this could be a shorter sequence within the template
Name two thermostable DNA polymerases
Taq Thermus aquaticus
Pfu Pyrococcus furiosus
Pwo Pyrococcus woesei
Phusion (human-made)
Expad HiFi, Expand 20kbplus
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?
it has no proofreading
can be used to generate random mutations
What is ‘subcloning’?
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
What aspect of Taq DNA pol makes it useful for some subcloning reactions such as GEM-T?
it leaves a 3’ A overhang
What major advantage does using Pfu and Pwo over Taq DNA pol in PCR have?
Pfu and Pwo have proof-reading and are therefore accurate which is important in order to generate faithful copies of template
Why does using Pfu or Pwo instead of Taq in PCR lead to lower efficiency?
They leave no overhangs, therefore blunt end cloning is required which has lower efficiency than 3’A overhang
In PCR primer design, what is the primer sequence complementary to?
the DNA adjacent to the region going to be amplified
Why must a PCR primer be ‘sufficiently long’? ANd what is the usual range for this length?
Long so that it only binds to the single site within the template DNA
18-30 nucleotides
What percentage must G and C content be for PCR primer design?
between 40%-60%
Which base must be present at the 3’end of primers for PCR?
G or C
Why must G or C be at the 3’ end on primers used for PCR?
GC clamp stabilises primer binding
What are inverted repeats?
A single stranded sequence of nucleotides which is followed by its reverse complement downstream (e.g. TTACGnnnnCGAA )
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?
Inverted Repeats (causes hairpin loops)
Define primer Tm
Primer melting temperature is the temperature at which half of the double stranded DNA will dissociate to become single stranded
How do you calculate primer Tm?
2(A+T) + 4(G+C)
What range of temperatures is common for primer Tm?
52-58 celsius
or 55-70 celsius
How do you calculate a rough value for annealing temperature (in PCR) using primer Tm?
usually Tm minus 5 Celsius
How do primers provide specificity? consider their structure
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
What buffer is used in PCR?
KCl, MgCl2
Why is a buffer needed in PCR?
They provide a suitable chemical environment for activity of DNA polymerase
WHat is the typical range for PCR buffer pH?
8.0-9.5
Why is K+ (potassium ions) from KCl a common component in PCR buffer when Taq DNA polymerase is used?
it promotes primer annealing
