The Polymerase Chain Reaction (PCR) Flashcards
What did the three-dimensional crystal strcture proposed by Watson and Crick in 1953 suggested?
How DNA could be replicated.
From who did the suggestion of DNA by Watson and Crick, confirmed experimentally?
By Meselson and Stahl.
When did Meselson and Stahl confirmed experimentally the suggestion of Watson and Crick about DNA?
In 1958.
What did the understanding of how DNA was replicated at the enzymatic level provide?
Opportunities to develop gene cloning and DNA sequencing.
When did the understanding of how DNA was replicated at the enzymatic level provide opportunities to develop gene cloning?
In 1970s.
What did gene cloning include?
DNA ligase.
Restriction enzymes.
What did DNA sequencing include?
DNA Polymerase.
Klenow fragment .
What did the modified version of Escherichia coli DNA Polymerase used to do?
Sequencing NDA.
End-labelling DNA.
Synthesizing larger fragments.
What was the Klenow fragment in 1970?
A modified version of the Escherichia coli DNA Polymerase.
What did Klenow needed?
A DNA template.
A primer.
Deoxynucleotide triphosphates.
What was the DNA template?
Single-stranded DNA.
What was a primer?
RNA/DNA.
What were the deoxynucleotide triphosphates?
dNTPs.
How was the Klenow characterised?
Very expensive.
Temperature-sensitive.
Rapidly degrading at the sequencing temperature of 37 degrees.
Storing poorly at 10 degrees.
What is the PCR?
The Polymerase Chain Reaction.
Who invented the PCR?
Kary Mullis.
When did Kary Mullis invented the PCR?
In 1983.
What is the function of PCR?
Reinforcing a specific region of DNA.
Of what did PCR became an alternative?
Of isolating specific genes of interest for cloning.
Where did the reinforcing of specific DNA region lead?
To development of the technique.
How did the reinforcing of specific DNA regions lead to development of the PCR technique?
Testing for presence/absence of a target sequence in complex samples in diagnostics and forensics.
What did PCR also provided?
An improved, more efficient Sanger sequencing.
What does PCR replicate?
The target DNA.
How does PCR replicate the target DNA?
By semi-conservative replication.
How many times is the semi-conservative replication of the target DNA by PCR repeated?
Many cycles.
Why is the semi-conservative replication of the target DNA by PCR repeated many cycles?
To produce huge DNA amounts.
How does each newly-synthesized strand of DNA act?
As a template.
Where does each newly-synthesised strand of DNA act as a template?
In the following replication cycle.
What is DNA production?
A logarithmic process.
What happens in the logarithmic process of DNA production ?
The number of copies of the target sequence increase by 2N where N is the number of cycles.
2N x Y.
Y = the number of target sequences.
What does PCR require?
Template DNA.
What does template DNA include?
An unbroken section.
What is the unbroken section of the template DNA?
The target sequence.
What do the 2 DNA oligonucleotide primers do?
Define the 2 ends of the target.
One maps the top strand.
One maps the bottom strand.
Which are the nucleotides in PCR?
Deoxyribonucleotide triphosphates: dATP. dCTP. dGTP. dTTP.
Which enzyme is used in PCR?
DNA Polymerase.
What else do we us in PCR?
PCR buffer.
BSA.
Why to we use a PCR buffer in PCR process?
To modify pH .
To provide Mg2+.
Why do we use BSA in PCR process?
To prevent non-specific protein-DNA interactions.
What does the PCR procedure use?
A set of three steps.
How many times are the 3 steps of PCR procedure repeated?
As many times as necessary.
Why do the 3 steps of PCR procedure repeated as many times as necessary?
To produce the required amount of target sequence.
Which are the 3 steps of PCR procedure?
- Denaturation.
- Annealing.
- Extension/Elongation.
What does each cycle generate?
New DNA strands.
How does each cycle generate new DNA strands in PCR procedure?
Via semi-conservative replication.
What happens when more cycles are completed in PCR?
More DNA is produced.
When does a new DNA Polymerase need to be added?
After every cycle.
Why does a new DNA Polymerase need to be added after every cycle?
Because Klenow fragment it is not thermally stable.
How was the early PCR procedure characterised?
Very expensive.
What are thermophiles?
Organisms.
Bacteria.
Archaea.
What do thermophiles do?
Thrive at relative high temperatures.
In which temperatures do thermophiles thrive?
41-122 degrees.
In what were biochemists initially interested?
In understanding how enzymes could still function at high temperatures without being degraded.
Where were the high-temperature enzymes used?
In biotechnology.
Paper making.
Washing powders.
How were DNA Polymerases from thermophiles than those form mesophiles, such as Escherichia coli, characterised?
More stable.
Less expensive.
Longer lasting.
What did the original PCR method required?
New Klenow to be added after every extension stage.
What did the requirements of the original PCR method made the procedure?
Slow.
Expensive.
From where was an alternative thermostable DNA Polymerase isolated?
From Thermus aquaticus by Chien et al.
When was an alternative thermostable DNA Polymerase isolated?
In 1976.
How could the alternative thermostable DNA Polymerase be used?
Without the constant need for replacement at the end of each cycle.
How did the alternative thermostable DNA Polymerase became known?
As ‘thermal cycling’ PCR.
How many times are the 3 stages of PCR cycles?
20-30 times.
Why are the 3 stages of PCR cycled 20-30 times?
To generate the required amount of product.
What happens in the Denaturation step of PCR?
Double stranded DNA is melted at high temperature to form single stranded template DNA.
What happens in the Annealing step of PCR?
The temperature is dropped –> primers bind to the target template DNA through complimentary base pairing.
What happens in the Extension process of DNA?
Polymerase binds to the primer/DNA complex –> adds nucleotides in the 5’-3’direction.
What do various methods allow?
The monitoring of the PCR.
What do techniques of PCR monitoring use?
DNA stains.
What happens in the techniques of PCR monitoring where DNA stains are used?
Only fluoresce when bound to DNA.
How are the techniques of PCR monitoring that use DNA stains called?
Real-time PCR. Quantitative PCR (qPCR).
How is the cycle at which product is first detected termed?
The quantitation cycle: Cq.
What do lower Cq valuesmean?
Higher initial copy numbers of the target.
What do higher Cq values indicate?
That fewer target copies were present in the sample.
For what are primers needed?
To define the ends of the target sequence.
Why are the primers needed to define the ends of the target sequence?
To be reinforced.
Where do PCR primers bind?
To the DNA sequences.
How do the PCR primers bind to the DNA sequences?
By base-pairing.
What do the PCR primers provide?
A 3’- OH.
For what do the PCR primers provide a 3’-OH?
For DNA Polymerase.
Why do PCR primers provide a 3’-OH for DNA Polymerase?
To begin synthesizing the new DNA.
What was the primer synthesis originally?
Very hard to do efficiently at a large scale.
How were the PCR primers characterised?
Very expensive to produce.
How can the primers be?
Long and specific.
Short and random.
Why can primers be short and random?
To reinforce random stretched of DNA.
Why can primers be long and specific?
For a particular gene.
What must primers have?
Sequence information about the start forward primer and end reverse primer of the DNA piece to be reinforced.
How many nucleotides long can primers be?
6-30 nucleotides long.
What are the long primers?
More specific.
How many times do short primers bind to a DNA sample?
Many times.
How are long primers characterised?
Unique.
On what is the annealing temperature dependant?
On the properties of the primer.
How is the annealing temperature usually referred to?
‘Tm’.
What is the annealing temperature?
The temperature required to melt the primer-DNA complex.
What are the primer melting temperatures (Tm)?
Temperatures where 50% of the primer-DNA template duplex will melt.
How is the Tm estimated?
Tm = 2 x (A + T) + 4 x (G+ C).
What is the equation of Tm?
Insensitive to changes in salt concentration that might occur in different PCR reactions.
How is the Tm equation characterised?
Not as accurate a might be preferred.
Where will poorly-designed primers lead?
To no amplification of the target sequence.
Miss-amplification.
Where will poorly-designed primers lead to miss-amplification?
Where other fragments are amplified.
What are the sues of PCR?
Cloning. Mutagenesis. Amplification. Detection and typing. Detection.
What are the cloning and mutagenesis uses of PCR?
Primer sequences are modified to introduce RE sites/mutations/link different fragments of DNA together.
Of what is amplification a use of PCR?
Of a target sequence for Sanger sequencing of separate or combined reactions.
Of what is detection and typing uses of PCR?
Of bacterial and viral pathogens.
Of what is detection a use of PCR?
Of small mutations (SNPs).
Larger mutations associated with disease.
Of multiple repeat loci as part of forensic profiling.
How many PCR amplification can be undertaken in one reaction?
More than 1.
For what can multiplexing be used?
To target multiple sequences.
Why can multiplexing be used to target multiple sequences?
To build-up DNA profiles.
Where are DNA profiles useful?
In assessing the likelihood of developing genetic disease.
In forensic cases.
What is DMD-associated dilated cardiomyopathy?
A form of heart disease.
Of what is dystrophin a part in skeletal and cardiac muscles?
Of a group of proteins that work together.
A protein complex.
Why do the proteins work together in disease diagnostics?
To strengthen muscle fibres.
Protect the muscle fibres from injury.
Why do proteins protect the muscle fibres from injury?
Because they contract and relax.
From what can disease result?
Deletion of one/more exons of the dystrophin gene.
What are the disadvantages of PCR?
The target sequence need to be known in order to design primers.
The PCR product is short < 500bp.
The number of cycles is limited as the polymerase looses efficiency over time and dNTPS and primers are used up.
PCR depends on a pure DNA sample.
Contaminants effect the polymerase.
PCR introduces mutations/errors into the amplified DNA.
PCR is expensive compared to other methods of DNA isolation.
What are the advantages of PCR?
Compared to other methods of isolating specific fragments of DNA, PCR is rapid and easier.
It can be used to produce very sensitive protocol/diagnostic assay.
It is robust and insensitive to contaminants and small changes in protocol.
It is flexible.
It is easily modified to suit different purposes.
DNA and RNA isolation and PCR reactions can be bought as part of quality-controlled kits with the appropriate internal controls that satisfy requirements for diagnostic and forensic applications.
