LECTURE 3 (PCR) Flashcards
(39 cards)
What’s the denaturing step of PCR?
break the pieces of DNA by rising the temperature to 92-95ºC because we need to separate both strands
What is the temperature we need to break two strands of DNA?
92-95ºC
What is the polymerization step?
You need to reduce the temperature down so the primers you add will hybridize to the complementary sequences and will allow the polymerase to start at those ds sequences.
What is the formula for the products of PCR? is it a linear formula?
Y=X2^n
This is an exponential formula, being Y the final amount, X the initial amount of DNA and n the number of cycles.
What is PCR used for? (3)
Either to amplify pieces of DNA, for screening cDNA libraries or analyze DNA to mutagenize plasmids
Is it a infinite exponential graph?
No. at certain point things happen in the reaction such as the degradation of the polymerase or decrease of [dNTP] that will stop the graph from being exponential
What is the usual polymerase used in PCR? Why? How was it discovered?
Taq polymerase discovered in thermal vents, the enzymes in this organism (thermophlius aquatics) have evolved to withstand very high temperatures , it lacks proofreading activity but has high processivity
How can we know the amount of DNA produced in PCR?
Simply by using a fluorescent dye e.g Sybr green
How can we tell the difference between two curves of PCR?
if the difference = 1 cycle then there was twice as much DNA in the one that enters in exponential phase the 1st.
What are the enzymes used in PCR?
They have to be thermostable enzymes because of the temperature changes and they can’t be easy to denature.
Thermal PCR cycle.
- Start Tª
- Tª for breaking the strands
- Tª of annealing
- extension phase Tª
- 4ºC (we don’t want the DNA to start too early to denature)
- 92-95ºC (we don’t want the water to boil out!)
- 55ºC
- 72ºC
What is the extension phase of PCR?
The elongation of the strains and occurs at 1000bp/min
What are the two temperatures that can vary in PCR?
The Tª for primers, which depends on the length and base composition of them
Tª of the extension phase which depends on the sample size and the polymerase used
What are thermal cycles?
Machines that allows the temperatures in PCR to vary safely. Current designs exploit Peltier based. It uses small tubes so the change of temperatures is very fast
Which polymerases are thermostable? i.e allow Tª up to 98ºC and retain activity for a long time.
Taq polymerase (Thermophilius aquaticus, found in bacteria in thermal vents)
Vent (Thermococcus litoralis)
Pfu (Pyrocuccus furiosus)
From how many cycles does PCR amplify rubbish?
30-35 cycles
Why isn’t Pfu the polymerase used for PCR since it has the lowest error rate?
Because it’s not the only thing taken into account, reliability is also a key thing. BUT principally because the higher the fidelity the slower the speed so we need to combine speed with accuracy.
What are the characteristics of polymerases (4)
- 5’-3’ exonuclease activity
- 3’- 5’ exonuclease acitivy
- error rate
- km with respect to nucleotides and DNA varies
What does an advert have to include?
- tell the accuracy
- the speed
- how big the size range of amplification is in kb
- show the fidelity
What is the application of cloning of PCR? (3)
- Amplify specific fragments of DNA from complex mixes
- Prepare specific fragments with ends (termini) that can be adapted for joining together
- Taq pol introduce targeted and random mutations
what is site-directed mutagenesis?
The introduction of designed mutations in a piece of DNA and formation of a primer.
How does site-directed mutagenesis work?
A high fidelity enzyme is used. You need to design primers as always but with the exception that they contain a seq that is the sequence you want to change therefore they will anneal with a sequence that won’t be exactly the same. After hybridization–> PCR. The producs will include the mutations and you can then transform a bacterium.
before we get the products we need to use a 4bp recognition RE e.g DpnI that is sensitive to dA methylation so it’ll cut only DNA methylalated at the A site (the template) and then DNA that is not methylated is amplified(the new strand won’t be methylated), after this, the products are ligated to obtain a primer.
who invented the PCR and when?
Mullis in 1985 ten years after Taq was found and the same year that it was found that elk genes are formed by introns and exons.
What is a limitation of PCR?
primers, because you have to know part of the sequence of the DNA fragment.
