PCR Flashcards

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1
Q

most commonly used polymerase in PCR reactions

A

Taq from Thermus aquaticus

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2
Q

Who developed PCR?

A

Kary Mullis and Michael Smith (1993)

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3
Q

Applications of PCR

A
  1. detection of DNA sequences
  2. molecular diagnosis of diseases
  3. DNA fingerprinting
  4. detection of viruses and bacteria
  5. phylogenetics
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4
Q

Two phases of PCR

A
  1. screening phase
  2. amplification phase
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5
Q

Give at least 5 considerations when designing a primer

A
  1. length should be 17-24 bases
  2. 40-60% GC content
  3. no formation of secondary structures
  4. should end with G or C but prevents runs of G and C
  5. annealing temperature should be the same for both primers and 5oC lower than the Tm
  6. 100% homology at 3’ end
  7. no self-hybridization
  8. %GC and Tm difference of 5 (or 2) between the two primers
  9. the %base composition should approximate that of the template
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6
Q

optimum length of primers

A

17-24 bases

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7
Q

what happens if the primer length is too short?

A

could anneal to more than one site

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8
Q

what happens if the primer length is too long

A

could not bind to any site

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9
Q

what happens if there are too much runs of Gs or Cs

A

mispairings

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10
Q

components of a PCR reaction mix

A
  1. buffer
  2. dNTP mix
  3. MgCl2
  4. primer pair
  5. DNA Pol (Taq pol)
  6. genomic DNA template
  7. sterile water
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11
Q

example of a PCR profile

A

Initial D - 95 - 5 mins
35 cycles
Denaturation - 95 - 1 min
Annealing - 55 - 1 min
Extension - 72 - 1 min
Final extension - 72 - 10 mins
Extended temp - 4

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12
Q

mechanism of RT-PCR

A

reverse transcription PCR
1. the poly-A tail of the mRNA is recognized by the oligo(dT) primer to form the mRNA/cDNA heteroduplex by reverse transcribing the mRNA using reverse transcriptase
2. RNase H or OH cleaves the mRNA, leaving the ss cDNA
3. DNA polymerase synthesized the complementary strand of the cDNA

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13
Q

difference between 5’ and 3’ RACE PCR

A

rapid amplification of cDNA ends PCR
for the 3’ RACE-PCR
- an oligo(dT) primer would anneal to the poly-A tail of the mRNA and will be reverse transcribed to form the cDNA
- a forward and reverse primer pair would be used to amplify part of the cDNA up to the poly-A tail, creating the second half of the cDNA

for the 5’ RACE-PCR,
- an anchor primer would anneal to the 5’ end of the cDNA after reverse transcription from the mRNA (and cleavage by the terminal transferase with oligo(dC)
- forward and reverse primers would amplify the part of the cDNA from the 5’ end, creating the first half of the cDNA

Joining of the products of these 2 RACE-PCR reactions can generate the full-length sequence of the cDNA

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14
Q

a dye used in qPCR that binds the minor groove

A

SYBR Green Dye

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15
Q

mechanism of the TaqMan probe

A
  • the primer and the TaqMan probe anneal to the complementary strand following denaturation
  • the intact TaqMan probe would have the reporter fluorophore and the quencher protein close, preventing emission of fluorescence
  • after hybridization and during elongation, Taq pol cleaves the probe and separates the quencher and reporter, generating fluorescence
  • detection of the fluorescence is used for quantification
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16
Q

the increment of fluorescent signal at each time point during PCR cycles in qRT-PCR

A

ΔRn

17
Q

PCR cycles in which a reporter fluorescent signal is accumulating but is not detected yet

A

baseline

18
Q

an arbitrary level of fluorescence chosen on the basis of baseline variability

A

threshold

19
Q

the cycle number at which fluorescence is first detected

A

Ct (cycle threshold)

20
Q

How is qRT-PCR used in differential gene expression?

A

The differences in Ct values from the resulting graphs would determine how much of the gene is present. Lower Ct values would mean that there is a higher amount of starting template, which means a higher amount of the gene in that sample.

21
Q

qRT-PCR results must be repeated when?

A

the resulting curves are invalid or had late amplification