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PCR Flashcards

1
Q

• Started in 1990 involving about
30 different institutions

A

THE HUMAN GENOME PROJECT

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2
Q

HUMAN GENOME PROJECT GOAL

A

• Goal: Fully sequence the entire human genome

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3
Q

THGP

• Initial completion:
• Mission Accomplished:

A

April 2003

MAY 2021

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4
Q

HUMAN GENOME

Toal size

A

~ 3.2 billion bp

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5
Q

Studying Specific Sequences
•_______ - the genome is LARGE
•_______ - amount of DNA
samples are VERY small

A

SPECIFICITY

AMPLIFICATION

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6
Q

Came upon a way to double his test target DNA by giving him 2^1 copies

Repeating N times would yield: 2^N

By adding an oligonucleotide primer with the 4 dNTPs and DNA polymerase in a chain reaction

A

Dr. Kary Mullis

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7
Q

Dr. Kary Mullis

______Nobel Prize Winner in
Chemistry for the_______

A

1993

Polymerase Chain Reaction

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8
Q

PCR STEPS (3)

A

1 Denaturation
2 Annealing
3 Extension

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9
Q

• Double-stranded DNA is separated into 2 single strands

A

Denaturation

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10
Q

DENATURATION

• temperature:
• Time:
• Larger template = longer time

A

94-96°C

several seconds to minutes

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11
Q

• MOST CRITICAL step for specificity

A

Annealing

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12
Q

Primers hybridize to their complementary DNA sequences

A

ANNEALING

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13
Q

ANNEALING

• Temperature:
• Starting point is determined using the_______

A

50-70°C

Tm of the primer sequences

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14
Q

Melting Temperature (Tm)

Formula 1 and 2

A

Tm = 81.5 deg C + 16.6 log M + 0.41(%GC) - 0.61% formamide) - (600/n)

Tm = 81.5 deg C + 0.41(%GC) - (675/n)

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15
Q

• Performed by DNA polymerase
• Polymerase synthesizes a copy of the template DNA
• Catalyzes formation of the phosphodiester bonds between dNTPs and the 3’ end of the primer

A

EXTENSION

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16
Q

EXTENSION

• Performed by______
• Polymerase synthesizes a copy of the______
• Catalyzes formation of the _______between dNTPs and the 3’ end of the primer

A

DNA polymerase

template DNA

phosphodiester bonds

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17
Q

EXTENSION

• Temperature:_______
• End result:______

A

68-72°C

2x template number

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18
Q

Temperature (°C)
Time (sec)

Denaturation

A

90-96
20-60

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19
Q

Temp
Time

Annealing

A

50-70
20-90

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20
Q

Temp
Time

Extension

A

68-75
10-60

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21
Q

• Single-stranded DNA fragments

A

PRIMERS

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22
Q

PRIMERS

• BP LENGTH?

A

20-30 bp long

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23
Q

• Works like the primers in vivo
• Determine the specificity of PCR
• Contain sequences complementary to sites flanking the region of interest

A

PRIMERS

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24
Q

• 5’ to the sequences amplified
• Hybridizes with the minus strand

A

• FORWARD PRIMER

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25
Q

• 3’ to the sequences amplified
• Hybridizes with the plus strand

A

• REVERSE PRIMER

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26
Q

• FORWARD PRIMER
•____ to the sequences amplified
• Hybridizes with the____ strand

A

5’

minus strand

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27
Q

• REVERSE PRIMER
•____ to the sequences amplified
• Hybridizes with the____ strand

A

3’

plus strand

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28
Q

PRIMER

• Binding to target is affected by: (2)

• _______of the forward and reverse primer must be similar

A

• Primer sequence (%GC)
• Length of strand

Melting temp (Tm)

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29
Q

DNA TEMPLATE
• From nucleotide extraction

• Routine clinical analyses requires:
______

A

100 ng - 1 ug

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30
Q

• Best templates (3)

A

• Good condition
• Free of contaminants
• No nicks and breaks

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31
Q

• Building blocks of DNA

• dNTPs - dATP (adenine), dTTP (thymine), dGTP (guanine), dCTP (cytosine)

A

DEOXYRIBONUCLEOTIDE BASES

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32
Q

DEOXYRIBONUCLEOTIDE BASES

• Usual requirement:

A

0.1-0.5 mM of each

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33
Q

DNA polymerase

• First polymerase from____
• Needed to be added after each round of_____

A

E. coli

denaturation

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34
Q

DNA polymerase

• Thermostable
• Good fidelity

A

• Taq polymerase
• Thermus aquaticus

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35
Q

DNA polymerase

• Also has reverse transcriptase activity
• Used in RT-PCR (RNA template)

A

• Tth polymerase
• Thermus thermophilus

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36
Q

• Provide optimal conditions for enzyme activity
• pH buffers and salts that provide cations

A

PCR BUFFER

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37
Q

• Provides pH for optimal enzyme activity and accurate amplification
• рН 8 - 9.5
• Can have accessory comoponents

A

Tris buffer

38
Q

Tris buffer

• Provides pH for optimal enzyme activity and accurate amplification
• рН____
• Can have accessory comoponents

39
Q

Binds inhibitors and stabilizes the enzyme.

A

Bovine serum albumin (10 to 100 m g/ mL)

40
Q

Provides reducing conditions that may enhance enzyme activity.

A

Dithiothreitol (0.01 mM)

41
Q

Lower the denaturing temperature of DNA with high secondary structure, thereby
increasing the availability for primer binding.

A

Formamide (1% to 10%)

42
Q

May also reduce secondary structure to allow polymerase extension through difficult areas.

A

Triton X-100, glycerol, and dimethyl sulfoxide (1% to 10%)

43
Q

Monovalent CATIONS

A

(KCl and (NH4)2SO4 )

44
Q

(KCl and (NH4)2SO4 )

• Affect denaturing and
annealing temperatures

A

Monovalent CATIONS

45
Q

• ↑ salt concentration = longer
DNA sequences denature
slower

A

MONOVALENT CATIONS

46
Q

• Mg2+ is needed by DNA pol
• 1 NTP = 1 Mg atom

• If too low (↓ )
• By EDTA/other chelators
• ↓ amplicons

• If too high (↑)
• ↑ nonspecific products

A

divalent CATIONS
(MgCl2)

47
Q

divalent CATIONS

• _____is needed by DNA pol
• 1 NTP = 1 Mg atom

• If too low (↓ )
• By EDTA/other chelators
• ↓ amplicons

• If too high (↑)
• ↑ nonspecific products

48
Q

Machine:
• First PCRs
• Multiple water baths or heat blocks
• Done manually

A

thermal cycler

49
Q

• Rapidly & automatically change to the required temperatures for each step and holds it there for designated periods

A

Thermal cyclers

50
Q

CONTROL

Enzyme is active
Buffer is optimal
Primers are priming
Machine is working

51
Q

CONTROLS

• Negative control without DNA
• “Contamination” control

A

Reagent Blank

52
Q

CONTROLS

Negative control with DNA lacking the target sequence

A

Negative template

53
Q

CONTROLS (3)

A

POSITIVE
REAGENT BLANK
NEGATIVE TRMPLATE

54
Q

• Balance between aggressive amplification and avoidance of contaminating template

A

Contaminants

55
Q

• Major cause of contamination:

• presence of….

• Can be resolved (2)

A

PCR products from previous amplifications

physically or chemically

56
Q

PHYSICAL:

• Decontamination of pre-PCR areas
• Induces base damage that catalyzes the formation of single- and double-stranded breaks in DNA
• Enhanced effectivity with the addition of psoralens

57
Q

PHYSICAL:

• Enhanced effectivity with the addition of______

58
Q

• Widely used method for
decontamination and workspace prep

• Frequently wiped on surfaces that come in direct contact with specimen material to remove most DNA contamination

A

CHEMICAL: 10% Bleach

59
Q

BLEACH SOLUTION

A

Chemical: dUTP-UNG system

60
Q

• Substitutes the dTTP for dUTP in the PCR reagent master mix
• Uracil-N-glycosylase (UNG) degrades any nucleic acid containing uracil
• Incubation period at 50°C for 2-15 min added before PCR amplification

A

Chemical: dUTP-UNG system

61
Q

Chemical: dUTP-UNG system

• Substitutes the dTTP for dUTP in the PCR reagent master mix
• Uracil-N-glycosylase (UNG) degrades any nucleic acid containing uracil
• Incubation period at___________ added before PCR amplification

A

50°C for 2-15 min

62
Q

Concerning the primer

• Aberrant binding of the primer
•Carries the primer sequence and becomes a target for the next amplifications

63
Q

Concerning the primer

• Artifact in PCR
• PCR products double the size of primers
• Primers binding to each other

A

PRIMER DIMERS

64
Q

______has some activity at room temperature (22-25°C)

Primers can bind sequences other than their exact complements on the target (low stringency)

65
Q

SOLUTION 1:______
• Good primer design
• Optimal amplification conditions

Solution 2:______
• Type 1: Reaction mixes are prepped on ice and placed in a prewarmed thermal cycler
• Type 2: Wax barrier
Wax separates the enzyme and templates from the primers
• Type 3: Sequestered enzymes
• Supplied in inactive form

A

Good Preparation

Hot-start PCR

66
Q

Product Clean-up

AGAROSE GEL ELECTROPHORESIS
• Resolving amplification products
• Agarose digestion with_____ or ____

A

B-agarase or with iodine incubation

67
Q

Product Clean-up

Residual component removal
• Using spin columns
•_____+______

A

Shrimp alkaline phosphatase
(SAP) + exonuclease I (Exol)

68
Q

PCR Modifications

More than one primer pair added to PCR that are primed simultaneously

A

Multiplex PCR

69
Q

Advantage: Many in one go!
Disadvantage: Inefficient & with cross-reactivity

A

Multiplex PCR

70
Q

Two pairs of primers used to amplity a single target in separate PCR programs

A

Nested PCR

71
Q

Advantage: Increased sensitivity & specificity
Disadvantage: Time-consuming

A

Nested PCR

72
Q

Starting material is RNA and converted to DNA by reverse transcriptase

Used in gene expression studies

A

“RT-PCR”

Reverse transcriptase por

73
Q

Detects how much of the target sequence is present usually by fluorescence

Useful in detecting gene copy numbers, viral load, tumor load, and effects of treatment

A

“rt-PCR”

REAL-TIME PCR

74
Q

• Replaced Ethidium bromide (EtBr)
• Highly specific and robust fluorescence
• Less toxic than EtBr
• Cheaper than Taq Man

A

SYBR Green I Assay

75
Q

• Fluorescence occurs once the dye is separated from the quencher
• The probe makes it more specific than SYBR green
• Produces higher quality results

A

TaqMan Assay

76
Q

Binds to the minus strand (template strand)

Located 5’ to the sequence to be amplified

A

Forward Primer

77
Q

Binds to the plus strand (complementary strand)

Located 3’ to the sequence to be amplified

A

Reverse Primer

78
Q

(dNTPs)

A

Deoxyribonucleotide Triphosphates

79
Q

are the building blocks of DNA that are incorporated into the new DNA strand during extension.

80
Q

Types of dNTPs in PCR:
• → Adenine (A)
• → Thymine (T)
• → Guanine (G)
• → Cytosine (C)

A

dATP (deoxyadenosine triphosphate)

dTTP (deoxythymidine triphosphate)

dGTP (deoxyguanosine triphosphate)

dCTP (deoxycytidine triphosphate)

81
Q

is the enzyme responsible for synthesizing new DNA strands by adding dNTPs to the 3’ end of the primer.

A

DNA polymerase

82
Q

First DNA Polymerase Used in PCR:
• Originally, ____was used.
• However, it was heat-sensitive, meaning it had to be added after each

A

E. coli DNA polymerase

83
Q

• Most commonly used DNA polymerase in PCR.
• Thermostable: Can withstand high temperatures (94-96°C) during denaturation.
• Good fidelity (accuracy) but lacks proofreading ability (may introduce errors).

A

Taq Polymerase (Thermus aquaticus)

84
Q

• Has reverse transcriptase activity, meaning it can synthesize DNA from an RNA template.
• Used in RT-PCR (Reverse Transcription PCR) to amplify RNA.

A

Tth Polymerase (Thermus thermophilus)

85
Q

• Maintains a stable pH range of 8.0-9.5, which is ideal for DNA polymerase activity.
• Ensures that DNA strands remain stable during amplification.
• Prevents pH fluctuations that could inhibit the enzyme.

A

Tris Buffer

86
Q

Monovalent Cations (KCl, (NH4)2SO4)

A

KCl (Potassium chloride)
(NH4)2SO4 (Ammonium sulfate)

87
Q

Contamination control methods fall into two categories:

  1. – Prevents contamination by separating areas, using UV light, and controlling workflow.
  2. – Destroys contaminant DNA using bleach or enzymatic degradation methods.
A

Physical Decontamination

Chemical Decontamination

88
Q

Separation of Pre-PCR and Post-PCR Areas

A

Physical Methods of Decontamination

89
Q

occurs when primers bind to non-target regions of the DNA, leading to nonspecific amplification.

A

Mispriming

90
Q

Causes of Mispriming

A
  1. Low stringency conditions
  2. Poor primer design
  3. Contaminant DNA

MolBio LEC (8 decks)

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