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AMB > LECTURE 3 (PCR) > Flashcards

LECTURE 3 (PCR) Flashcards

1
Q

What’s the denaturing step of PCR?

A

break the pieces of DNA by rising the temperature to 92-95ºC because we need to separate both strands

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2
Q

What is the temperature we need to break two strands of DNA?

A

92-95ºC

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3
Q

What is the polymerization step?

A

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.

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4
Q

What is the formula for the products of PCR? is it a linear formula?

A

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.

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5
Q

What is PCR used for? (3)

A

Either to amplify pieces of DNA, for screening cDNA libraries or analyze DNA to mutagenize plasmids

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6
Q

Is it a infinite exponential graph?

A

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

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7
Q

What is the usual polymerase used in PCR? Why? How was it discovered?

A

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

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8
Q

How can we know the amount of DNA produced in PCR?

A

Simply by using a fluorescent dye e.g Sybr green

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9
Q

How can we tell the difference between two curves of PCR?

A

if the difference = 1 cycle then there was twice as much DNA in the one that enters in exponential phase the 1st.

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10
Q

What are the enzymes used in PCR?

A

They have to be thermostable enzymes because of the temperature changes and they can’t be easy to denature.

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11
Q

Thermal PCR cycle.

  • Start Tª
  • Tª for breaking the strands
  • Tª of annealing
  • extension phase Tª
A
  • 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
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12
Q

What is the extension phase of PCR?

A

The elongation of the strains and occurs at 1000bp/min

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13
Q

What are the two temperatures that can vary in PCR?

A

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

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14
Q

What are thermal cycles?

A

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

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15
Q

Which polymerases are thermostable? i.e allow Tª up to 98ºC and retain activity for a long time.

A

Taq polymerase (Thermophilius aquaticus, found in bacteria in thermal vents)
Vent (Thermococcus litoralis)
Pfu (Pyrocuccus furiosus)

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16
Q

From how many cycles does PCR amplify rubbish?

A

30-35 cycles

17
Q

Why isn’t Pfu the polymerase used for PCR since it has the lowest error rate?

A

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.

18
Q

What are the characteristics of polymerases (4)

A
  • 5’-3’ exonuclease activity
  • 3’- 5’ exonuclease acitivy
  • error rate
  • km with respect to nucleotides and DNA varies
19
Q

What does an advert have to include?

A
  • tell the accuracy
  • the speed
  • how big the size range of amplification is in kb
  • show the fidelity
20
Q

What is the application of cloning of PCR? (3)

A
  • 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
21
Q

what is site-directed mutagenesis?

A

The introduction of designed mutations in a piece of DNA and formation of a primer.

22
Q

How does site-directed mutagenesis work?

A

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.

23
Q

who invented the PCR and when?

A

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.

24
Q

What is a limitation of PCR?

A

primers, because you have to know part of the sequence of the DNA fragment.

25
Q

Why doesn’t water evaporate in PCR?

A

because the temperature doesn’t reach 100ºC and because the little tubes have a lid with either oil on top (not used anymore) or a heated lid that maintains a cte temperature throughout the tube.

26
Q

How should we design a primer? How long are they?

A

the more Gs and Cs the stronger it’ll bound to to seq because it uses 3 Hydrogen bonds. they are +/- 200 bp long. AVOID PLASMID COMPLEMENTARITY.

27
Q

Mg2+ in PCR?

A

yes, it can be used to optimize it increases the amplicon yield but decreases the fidelity

28
Q

How do we analyze the PCR products?

A

separate them into an agarose gel to ensure that only one fragment is amplified this is accepted if the molecular weight is all the same in all the strains. Then we can either sequence or blot to make sure we have a good sequence.

29
Q

What is nested PCR?

A

Increases the specifity of PCR and reduces non-specific bindings. It involves two sets of primers. (diagram in pink)

30
Q

Why are PCR products used for cloning if they’re blunt ended?

A

Because Taq pol leaves a little A overhang at the end allowing TA cloning.

31
Q

What is qPCR?

A

quantitative PCR or real-time PCR. It is the analytical method for quantifying target DNA.

32
Q

Description of qPCR

A

We need to measure the amount of fluorescence e.g Sybr green (fluoresces when bound to dsDNA) which is proportional to the [DNA] , the amount of DNA is related to the Ct value, the difference bt Ct would be proportional to the difference of the templates The HIGHER the [DNA] the LOWER the Ct value. You need to set a threshold and measure the cycle at which that threshold is reached and that is the Ct (critical threshold). The Ct value is the point from which all the analysis begins.

33
Q

Which are the two ways of analyzing the data of qPCR?

A
  • The absolute method; compares the Ct value of the sample with a standard curve and determines the original [DNA]
  • Relative method: uses reference genes to see the difference in gene expression bt two treatments. Do PCR on both samples + reference genes then find the difference in Ct values the ratio bt deltaCt tells how much gene expression has changed.
34
Q

What is the Taqman method?

A

Here instead of Sybr Green, a probe is used. this probe binds to the product and has a fluorescent particle at the 5’ end and a quencher at the 3’end. the exonuclease activity of taq pol will cut off the fluorescence particle from the quencher therefore fluorescence is shown and it can be measured the same way.

35
Q

What is RT PCR?

A

It’s not real time PCR, it’s reverse transcriptase PCR. A combination of RT with PCR using mRNA and getting cDNA. mRNA might be contaminated so make sure we don’t use primers that bind to DNA.

36
Q

Difference between RT PCR and qPCR

A
  • qPCR; measures the initial amount of DNA or of the final products by using fluorescent probes; quantitatively method
  • RT PCR: creates cDNA and detects the gene expression, qualitatively method
37
Q

What’s better RT PCR or qPCR

A

neither of them, it depends on the aim of the experiment but a combination of both q RT PCR would be the best tool for detecting RNA levels

38
Q

What are overall the 4 main applications for PCR and what is not an application?

A
  • Amplifying DNA from ancient tissues to reveal information about prehistoric societies
  • Mutagenising plasmids
  • Screening cDNA and DNA libraries
  • Detecting DNA in small quantities of samples from suspects at crime scenes
    is NOT used to synthesize large amounts of DNA for commercial purposes
39
Q

RACE PCR

A

Rapid Amplification of cDNA Ends in PCR: its used to obtain full length cDNA and extend the fragments known

AMB (12 decks)

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