PCR Flashcards
What does PCR stand for?
Polymerase chain reaction
Why is PCR needed if molecular cloning exists?
- May not be enough DNA
- DNA may be in with lots of other DNA molecules
What is the basic process of PCR?
To amplify a specific piece of DNA
Explain how PCR is specific
will only get amplification of your selected sequence
Explain how PCR is selective
it can amplify a specific sequence from a mixture of DNA sequences
Describe how there is an exponential growth when PCR is used?
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
How many is the typical number of PCR cycles that takes place?
approx. 30
What are the 3 stages of PCR?
- Denaturation
- Primer annealing
- Primer extension
At what temperature does denaturation occur?
95 degrees Celsius
At what temperature does primer annealing occur?
55-65 degrees Celsius
At what temperature does primer extension occur?
68-72 degrees Celsius
What occurs during the denaturation stage?
Double-stranded DNA dissociates into single-stranded DNA, which is facilitated by the high temperatures seen at this stage.
What occurs during the primer annealing stage?
Primers bind to the complementary sequence on ssDNA (single-stranded DNA). Primer binding is antiparallel.
Is primer annealing parallel or antiparallel?
antiparallel
What occurs during the primer extension phase?
DNA polymerase synthesises new strands of DNA from the 3’ end of the primers.
Why is the process of PCR considered a semi-conservative process?
It is made up of a new strand of DNA & another of old DNA.
What are the 7 things needed for PCR?
- 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
Why is it necessary to have primers for PCR?
Polymerase requires a free 3’ OH to start. As a result we need to know what the sequence is
Why is it necessary to have deoxyribonucleoside triphosphates (dNTPs) in PCR?
They are the DNA bases to make the new DNA strand
Why is it necessary to have a buffer in PCR?
PCR requires the correct pH & ions
What ion is commonly used in PCR?
magnesium chloride
Why is temperature important in PCR?
each PCR cycle consists of stages that each need a specific temperature
Why are thermostable polymerases used from thermophilic organisms during PCR?
otherwise there is a danger that the high temperatures experienced in PCR at certain points may denature the DNA polymerase
What 4 properties are important in DNA polymerases used in PCR?
- thermostability
- extension rate
- processivity
- fidelity
Why is thermostability important in DNA polymerase used in PCR?
There are high temperatures associated with PCR
What is extension rate, when discussing DNA polymerase used in PCR?
how fast the DNA polymerase can replicate a template
What is processivity, when discussing DNA polymerase used in PCR?
how often the DNA polymerase falls off & has to re-associate
What is fidelity, when discussing DNA polymerase used in PCR?
how accurate it is (a higher fidelity would mean that less mutations were introduced into the PCR products
What are the 2 main DNA polymerases?
Taq & Pfu
What are the advantages of using Taq as a DNA polymerase?
- rapid extension
- high processivity (efficiency)
- adds an adenine overhang
What are the disadvantages of using Taq as a DNA polymerase?
- no proof-reading activity
- low fidelity (accuracy)
What are the advantages of using Pfu as a DNA polymerase?
- high fidelity (accuracy)
- very thermostable
What are the disadvantages of using Pfu as a DNA polymerase?
- low processivity (efficiency)
- products are blunt-ended
- lower extension rate
What are the 4 main important properties of PCR primers?
- minimum size for specificity
- specific to your template
- come in pairs
- appropriate melting temperature
What is the minimum size of a PCR primer for it to provide specificity?
17 base pairs is an absolute minimum - usually around 20 base pairs.
What will occur if there is only one PCR primer?
Only one strand will be replicated.
What is the melting temperature (Tm)?
the temperature at which the primer will dissociate from the DNA template
What is the usual range of melting temperatures of PCR primers?
60-64 degrees Celsius
What temperature should the annealing temperature be (relative to melting temperature)?
5 degrees Celsius lower than the melting temperature
What is the name given to the temperature at which 50% of PCR primer is annealed & 50% is not?
Tm = melting temperature
What is the consequence of the melting temperature being too high?
self-annealing may occur
What is the consequence of the melting temperature being too low?
primer often isn’t very specific
What occurs if the annealing temperature is too low?
the primers may non-specifically bind to other DNA sequences
What occurs if the annealing temperature is too high?
the primers may not bind efficiently (or at all) reducing product yield
What is the old fashioned way of calculating the primer melting temperature (Tm)?
(2+4)
+ 4 degrees Celsius per G/C
+ 2 degrees Celsius per A/T
What is the modern way of calculating Primer Tm?
use software programmes to calculate the most suitable temperature
What is the problem with cloning using PCR?
- 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
How can we clone a PCR product?
by ligating a PCR product into a vector
What problem may evolve as a result of trying to put a PCR product into a vector?
The insertion may be in the wrong orientation
What can be done to primers, to aid in the insertion of PCR products into vectors?
there can be restriction enzymes inserted into the primers
What can be done to prevent the PCR products being inserted into the vector in the wrong orientation?
Use 2 different primers
What is an example of a restriction enzyme on a forward primer?
EcoRI
What is an example of a restriction enzyme on a forward primer?
BamHI
What are the advantages of incorporating restriction enzymes into primers?
- more efficient ligation (sticky ends after restriction digest & the vector cannot ligate to itself
What are 2 variants of PCR?
- reverse PCR (PT-PCR)
- quantitative PCR (qPCR)
What is an overview of reverse PCR?
- RNA is reverse transcribed into cDNA.
- PCR used to amplify specific cDNA sequence
What is cDNA?
complimentary DNA
What are the potential uses of RT-PCR?
- molecular cloning of protein coding cDNA sequence
- analysis of RNA sequence/expression
What happens to the first strand in RT-PCR?
- reverse transcriptase synthesis of first strand of DNA.
- Poly (dt) primers bind poly(A) tail of mRNA.
- RNA is removed
What happens to the end of the first strand during reverse transcription PCR?
Loops back to form the start of another strand - forming a hairpin
What fragment synthesizes the second strand during RT-PCR?
Klenow fragment of DNA polymerase I
What is special about the Klenow fragment?
Retains useful polymerase function, however it has lost 5’ - 3’ exonuclease activity.
What is the ct value in quantitative PCR?
When the fluorescence generated by the DNA produced exceeds over the background
What is the fluorescent dye used in qPCR?
SYBR Green
When does the fluorescent dye SYBR Green fluoresce?
Fluoresce when bound to double-stranded DNA
What can be attached to one end of a fluorescent probe to prevent fluorescence from occurring?
Quencher
When does the quencher on a fluorescent probe get removed?
when the template gets replicated, the polymerase chops it off
What does the actual letters ‘Ct’ stand for?
cycle threshold
Why is qPCR still useful even though it can’t accurately measure the amount of DNA present?
The Ct value can be used as a relative measure to compare different DNA strands
What is the equation of calculating the fold difference of the Ct value?
2 (to the power) - delta CT
What is the most accurate way of finding out the fold difference?
2 (to the power) - delta delta Ct
What equations is fold difference made up of?
delta delta Ct = delta Ct A - delta Ct B
What is the equation for delta Ct A?
delta Ct A = Ct A test - Ct A ref
What is the equation for delta Ct B?
delta Ct B = Ct B test - Ct B ref
