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Applied Genetics > Polymerase Chain Reaction > Flashcards

Polymerase Chain Reaction Flashcards

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1
Q

what does PCR produce

A

millions of copies of a target sequence from template DNA in a few hours, compared with bacterial cloning which takes longer and is less efficient

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2
Q

what can PCR be used for

A
  • Generation of hybridization probes
  • DNA cloning
  • DNA sequencing
  • Isolation of DNA for recombinant technologies
  • Rapid screening of colonies
  • Genetic fingerprinting
  • Paternity testing and evolutionary relationships between organisms
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3
Q

what is a primer (or oligonucleotide)

A

strand of nucleic acid that functions as the starting point for DNA synthesis; in vivo the primer is made of RNA

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4
Q

what is DNA polymerase

A

enzyme that is involved in polymerization of deoxyribonucleotides into DNA. The new synthesised strand is complementary to the template strand

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5
Q

what does PCR reaction require

A
  1. ds DNA template
  2. A heat-resistant DNA polymerase
  3. Four nucleotides: dATP, dTTP, dCTP and dGTP
  4. Two short single-stranded DNA molecules serving as primers and complementary to each one of the strands of DNA
  5. Magnesium ions (act as cofactors for DNA polymerase)
  6. Buffer containing salt (DNA is acidic)
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6
Q

what are the steps in PCR

A
  1. Heat (94-99°C) to denature DNA strands
  2. Cool (50-65°C) to anneal primers to template
  3. Warm (68-72°C) to activate Taq polymerase, which extends primers and replicates DNA
  4. Repeat multiple cycles
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7
Q

how does DNA template denature

A

heat cause DNA strands to separate

occurs at 94 degrees C

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8
Q

what is annealing

A

pairing of DNA or RNA by hydrogen bonds to a complementary sequence to form a double-stranded polynucleotide

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9
Q

what are occurs during annealing

A
  • Primers bind to the template sequence at 60 degrees

- Taq polymerase binds to double-stranded substrate

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10
Q

what happens at 72 degrees

A

Taq Polymerase extends the primer

DNA is replicated

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11
Q

when is the exact target product made

A

in third cycle of PCR

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12
Q

what happens in cycle 1 of PCR

A

yields two molecules

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13
Q

what happens in cycle 2 of PCR

A

yields four molecules

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14
Q

what happens in cycle 3 ofPCR

A

yields eight molecules: 2molecules match the target sequence

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15
Q

what is Taq polymerase

A

Heat resistant – enzyme from Thermus aquaticus (living in hot springs) and withstands heat ≽ 100oC

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16
Q

what is the error rate of replication fidelity of taq polymerase

A

error rate 1/10000 nucleotides –> produces 16% mutations in DNA segment of 1kb

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17
Q

what is the replication of fidelity of taq polymerase

A

fast –> can amplify 1kb strand of DNA in ~ 30 secs at 72 degrees C

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18
Q

what does the taq polymerase lack

A

lacks 3’ to 5’ exonuclease proofreading mechanism

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19
Q

what is pfu polymerase

A

Heat-resistant, hyperthermophilic, has been isolated from the archaeon Pyrococcus furiosus and withstands heat > 100oC

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20
Q

what is the error rate of replication fidelity of pfu polymerase

A

proofreading activity (error rate of 1/1.3 million bp and yields only 2.6% mutations in a DNA segment of 1kb) because it has a 3’ to 5’ exonuclease mechanism which corrects mis incorporated nucleotides –> lower errors –> high

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21
Q

what is a disadvantage of pfu polymerase

A

Slower –> requires 1–2 mins to amplify

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22
Q

what does pfu produce and what what is required

A

1kb of DNA at 72° C fidelity

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23
Q

what can provide best characteristics for PCR

A

Mixing both Taq polymerase + Pfu polymerase it is possible to obtain both the fidelity of Pfu and the speed of Taq

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24
Q

how many base pairs should primers be

A

15 bp long

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25
Q

how much of the primers is made up of GC

A

40-60%

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26
Q

what temperature do primers anneal at

A

50-60 degrees C

27
Q

what temperature should forward and reverse primers anneal

A

approx same temperature

28
Q

what is the primer sequence

A

they should be unique(if a primer matches multiple sequences multiple products will result)
should not have self-annealing regions in their sequences (or will anneal to each other, so will not anneal to DNA want to amplify)
should not have self-annealing regions in their sequences (hairpin and foldback loops can be generated)

29
Q

what will happen is the pairs of primers anneal

A

they will form primer dimers

30
Q

what are the PCR types

A 
DNA PCR
RNA PCR
Multiplex PCR
In situ PCR
Real time PCR
31
Q

what is DNA PCR

A

amplification from DNA

32
Q

what is RNA PCR

A

amplification from RNA via Reverse Transcriptase reaction followed by DNA PCR

33
Q

what is multiplex PCR

A

different primer combinations simultaneously to detect & differentiate agents

34
Q

what is in situ PCR

A

localization of gene/ transcript

35
Q

what is real time PCR

A

real-time detection using various dye chemistries

36
Q

what is the difference in conventional and real time PCR detection

A

C - end point detection

RT - exponential point detection

37
Q

what is the difference in conventional and real time PCR for precision

A

C - poor precision

RT - high precision

38
Q

what is the difference in conventional and real time PCR sensitivity

A

C - low

RT - high

39
Q

what is the difference in conventional and real time PCR automation

A

C - non automated

RT - highly automated

40
Q

what is the difference in conventional and real time PCR quanification

A

C - difficult to quantify

RT - quantification

41
Q

what is the difference in conventional and real time PCR discrimination

A

C - Discrimination based on size

RT - Discrimination based on labeled probes, Tm

42
Q

what is the difference in conventional and real time PCR safety

A

C - carcinogenic

RT - safe

43
Q

what is the difference in conventional and real time PCR

A

C - post-PCR processing

RT - closed format, results and analysis obtained in real-time

44
Q

where is real time detection

A

in beginning of cycle is exponential

45
Q

where is the conventional PCR detection

A

in plateau area

46
Q

what is real time PCR able to detect

A

two-fold change (i.e. 10 Vs. 20 copies)

47
Q

what are the realtime PCR chemistries

A

DNA binding dyes

Probe-based

48
Q

what are the DNA binding dyes

A

SYBR green

Bebo

49
Q

what are the probe based

A

TaqMan

Molecular beacon

50
Q

what is SYBR green dye used for

A

identify real time

51
Q

what does SYBR do

A

will bind to any double-stranded DNA molecule, while the 5’ Nuclease assay is specific to a pre-determined target
attaches any dsDNA including primer-dimers

52
Q

what increases the SYBR green sye signal

A

more ds amplicons

53
Q

what happens in SYBR green and polymerization

A
  • DNA is denatured, the SYBR Green dye floats free
  • Extension phase begins as primers anneal
  • SYBR Green dye binds to the ds products / amplicons and fluoresces
54
Q

what is the SYBR green real time PCR

A

critical parameters - threshold value

melting temperature

55
Q

what is the threshold value

A
  • threshold line is the level of detection (set in the exponential phase of the amplification)
  • cycle threshold, Ct
56
Q

what is the melting temperature (Tm)

A
  • Temperature at which 50% DNA is denatured

- Only required for SYBR Green assay to check specificity

57
Q

what is the cycle threshold

A

directly proportional to amount of starting template

58
Q

how can the concentration of unknown sample be found

A

comparing Ct to a standard curve generated by dilutions of known amounts of a product

59
Q

what happens there is a high starting amount of nucleic acid

A

Higher the starting amount of the nucleic acid target, the sooner a significant increase in fluorescence is observed

60
Q

what is the melting curve analysis influenced by

A

Tm of a specific amplification is influenced by:

  1. length of the Amplicon
  2. Nucleotide sequences of the Amplicon (GC/AT ratio)
  3. PCR conditions
61
Q

what is a TaqMan probe

A

hydrolysis probes designed to increase the specificity of real-time PCR assays

62
Q

what happens in TaqMan probe use to the dye

A

Reporter dye’s emission is suppressed by the Quencher dye

63
Q

what happens when the enzyme reaches the TaqMan probe

A

the enzyme 5’ exonuclease activity begins

64
Q

what happens after exonuclease activity begins in TaqMan probe use

A

Reporter starts to fluoresce as it is separated from the Quencher

Applied Genetics (20 decks)

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