OVERVIEW OF PCR

studied byStudied by 9 people
5.0(1)
get a hint
hint

Polymerase Chain Reaction

1 / 97

encourage image

There's no tags or description

Looks like no one added any tags here yet for you.

98 Terms

1

Polymerase Chain Reaction

The first specific amplification method of any type. It is also the first and prototypical method for target nucleic acid amplification.

New cards
2

Kary Mullis

He discovered PCR in the mid-1980s, which he was granted a nobel prize for in 1993.

New cards
3

Polymerase Chain Reaction

It is a copy machine for DNA that revolutionized molecular biology.

New cards
4

False.

PCR is used to determine and confirm cellular or organismal presence by the detection of its genomic DNA or RNA (bacterial, viral, eukaryotic organisms).

Modified True or False.

PCR is used to determine and confirm cellular or organismal presence by the detection of its genomic RNA (bacterial, viral, eukaryotic organisms).

New cards
5

True

Modified True or False.

PCR is used to characterize cells or organisms by determining the DNA/RNA sequence of an amplified DNA/RNA target

New cards
6

Amplicons

These are the copies of a specific DNA sequence. It is formed after PCR.

New cards
7

In vitro

PCR is DNA replication ___ ___

New cards
8

DNA Polymerase

What is the main replication enzyme?

New cards
9

Base Pairing Rule (A-T, G-C)

DNA replication follows what rule?

New cards
10

False.

By amplifying the amount of nucleic acid materials obtained from clinical samples. Molecular techniques can effectively provide a quantitative representation of an individual's health

Modified True or False.

By amplifying the amount of nucleic acid materials obtained from clinical samples. Molecular techniques can effectively provide a qualitative representation of an individual's health

New cards
11
  • Water

  • Buffer

  • DNA Template

  • Thermostable Polymerase

  • Primer Pair

  • Magnesium (Mg2+)

  • Deoxynucleotide triphosphates (dNTPs)

What are the components of PCR?

New cards
12

Water

It is the medium for all other components.

New cards
13
  • Purified

  • Double distilled

  • Deionized

  • Autoclaved

  • Nuclease free

  • No detectable amounts of nucleic acid

Describe the water used for PCR.

New cards
14

Buffer

It stabilizes DNA polymerase, DNA, and nucleotides.

New cards
15

DNA Template

It contains the target region of interest.

New cards
16
  • Thermostable polymerase

  • Taq polymerase

  • It is used to catalyze DNA-dependent DNA synthesis.

  • Give an example

New cards
17

Thermus aquaticus

Taq polymerase comes from what thermophilic bacteria?

New cards
18

Primer Pair

These are oligodeoxynucleotides that flank the region of interest.

New cards
19

Forward Primer Pair

A type of primer pair that locates the replication start point along the template’s sense strand.

New cards
20

Reverse Primer Pair

A type of primer pair that marks a second replication start point along the antisense, downstream of its opposite primer.

New cards
21

18-30 base nucleotides

The length of a primer pair should be?

New cards
22

Magnesium

It acts as a cofactor for polymerase activity. It also stabilizes the DNA double helix.

New cards
23
  • Too little: Enzymes will not work

  • Too much: DNA will become extra stable and will cause non-specific priming

What are the consequence(s) if Magnesium is:

  • Too little

  • Too much

New cards
24

dNTPs

These are the equimolar building blocks of DNA.

New cards
25

Pre-PCR → PCR → Post-PCR

What is the typical PCR set-up in a laboratory?

New cards
26
  • Isolation of nucleic acid from samples

  • Preparation of PCR reaction components (mastermix)

  • Addition of nucleic acid template into the PCR Reaction Mastermix

Pre-PCR includes?

New cards
27
  • PCR

  • Denaturation, Annealing, Elongation

  • This PCR set-up includes Thermal Cycling

  • What is it composed of?

New cards
28

40

How many cycles can be done in thermal cycling?

New cards
29

Post-PCR

It includes the analysis of amplified DNA.

New cards
30

Pre-PCR

It is done prior to performing Molecular Biology techniques. It is the first step in PCR analysis.

New cards
31
  • Cellular lysis

  • DNA / RNA extraction

Sample treatment requires?

New cards
32

Heat denaturation

It refers to the high heat used to disrupt and separate DNA duplexes.

New cards
33

Heat

It breaks the hydrogen bonds of DNA template and separates into single strands.

New cards
34

94 C

DNA is heated to a minimum of (what temperature)?

New cards
35

Primer annealing

It refers to the attachment of primers to the template strand.

New cards
36

50 - 60 C

The DNA is cooled down to (what temperature)?

New cards
37

Individual ssDNA

DNA primers bind to ___ in annealing phase.

New cards
38

Primer extension

It refers to the elongation of daughter strand with DNA polymerase.

New cards
39

72 - 74 degrees C

What is the temperature in elongation?

New cards
40
  • Repetitively

  • Turnaround time

The PCR cycle would continue ___ in a predetermined ___.

New cards
41

Running the correct controls

It is essential for ensuring and maintaining the accuracy of the assay.

New cards
42

Positive controls

It ensures that the enzyme is active, the buffer is optimal, the primers are priming the right sequences, and the thermal cycler is cycling appropriately.

New cards
43

Negative control without DNA

It ensures that the reaction mix is not contaminated with template DNA or amplified products from a previous run.

New cards
44
  • Contamination control

  • Reagent blank

A negative control without DNA is also called?

New cards
45

Negative control with DNA

It lacks the target sequence (negative template control) and ensures that the primers are not annealing to nontarget sequences of DNA.

New cards
46
  • Gel electrophoresis

  • Capillary Electrophoresis

  • Nucleic acid hybridization

  • DNA sequencing

  • Real-time quantitative PCR

In Post-PCR, what are used to analyze PCR products?

New cards
47

False.

Depending on the application, the size, presence, or intensity of PCR products is observed after electrophoresis.

Modified True or False.

Depending on the application, the size, presence, or intensity of PCR products is observed before electrophoresis.

New cards
48
  • Reverse Transcriptase PCR (RT-PCR)

  • Multiplex PCR

  • Real-time Quantitative PCR

The modifications of traditional PCR

New cards
49

Reverse Transcriptase PCR

It refers to the conversion of the RNA template into its complementary DNA strand (cDNA), which is an essential step in the analysis of gene transcripts.

The cDNA is sequenced, cloned, and applied to estimate the copy number of specific genes in order to characterize and validate gene expression.

New cards
50

RNA virus

The enzyme reverse transcriptase comes from what organism?

New cards
51

Multiplex PCR

It is a powerful technique that enables amplification of two or more products in parallel in a single reaction tube. It typically employs different primer or probe pairs in the same reaction for simultaneous amplification of multiple targets.

New cards
52

Human Papilloma virus

What organism can be used in multiplex PCR?

New cards
53

Multiplex PCR

It is used widely in genotyping applications and multiple areas of DNA testing in research, forensic, and diagnostic laboratories.

New cards
54

Multiplex PCR

It is used for the detection of multiple pathogens using multiple primer sets in a single reaction.

New cards
55

Ladder

knowt flashcard image
New cards
56

Ladder

It is used as a standard to compare the bands from the gel electrophoresis.

New cards
57

Real time / Quantitative PCR

In this PCR, the accumulation of amplification product is measured as the reaction progresses with product quantification after each cycle.

New cards
58

Real Time / Quantitative PCR

Viral load of Hepatitis B virus is acquired through?

New cards
59
  • Early: high amount of starting template

  • Late: low amount of starting template

In Real Time or Quantitative PCR:

The detectable fluorescence in earlier cycles of the amplification program indicates ____, while fluorescence in later cycles indicates ____.

New cards
60
<p>Isolate DNA</p>

Isolate DNA

Quantitative real time PCR first step

New cards
61
  • Opaque

  • Fluorescent dye can be easily degraded in transparent PCR tube due to misdirection

  • What PCR tube is used for fluorescent dyes?

  • Why?

New cards
62

Centrifugation

A procedure that can remove bubbles.

New cards
63

Treshold cycle (CT)

The PCR cycle at which sample fluorescence crosses the threshold is the?

New cards
64

Fluorescent reporter molecule

Real-time detection of PCR products is enabled by the inclusion of a ____ ____ ____ in each reaction well that yields increased fluorescence with increasing of product DNA.

New cards
65

Ethidium bromide

The first approach in qPCR utilized ___, which is specific to double-stranded DNA. However, due to its toxicity, it was replaced.

New cards
66
  • SYBR Green

  • Specific, robust fluorescence, reduced toxicity

  • Ethidium bromide is replaced by ___. It is also specific with dsDNA.

  • What is its advantage?

New cards
67

Carcinogen

Ethidium bromide is a known?

New cards
68

Probe

Its purpose is to identify one or more sequences of interest within a large amount of nucleic acid.

New cards
69

False.

The probe should hybridize specifically with the target DNA or RNA that is to be analyzed.

Modified True or False.

The probe can either hybridize or neutralize the target DNA or RNA that is to be analyzed.

New cards
70
  • Taqman

  • Molecular Beacons

  • Scorpions

  • Fluorescent Resonance Energy Transfer

What are the common probes used in qPCR?

New cards
71

TaqMan

It was developed from one of the first probe-based systems for quantifying cDNA by qPCR.

New cards
72

Molecular Beacons

It measures the accumulation of product at the annealing step in the PCR cycle. It does not elongate or extend.

New cards
73

Scorpions

It is a target-specific primer which are tailed at the 5' end with a sequence complementary to part of the internal primer sequence, a quencher, a stem-loop structure, and a 5' fluorophore.

New cards
74

Fluorescent Resonance Energy Transfer (FRET)

It utilizes two specific probes, one with a 3' fluorophore (acceptor) and the other with a 5' catalyst for the fluorescence (donor).

New cards
75

Acceptor

The 3’ Fluorophore in FRET

New cards
76

Donor

The 5’ Catalyst of fluorescence in FRET

New cards
77

FRET

This PCR transfers energy.

New cards
78

True

Modified True or False.

Real-time PCR results can either be qualitative (the presence or absence of a sequence) or quantitative (copy number).

New cards
79

TaqMan probe

It hybridizes to the target sequences between the PCR primer-binding sites. The probe is covalently attached to a fluorescent reporter dye (R) at the 5' end and a quencher (Q) at the 3' end.

New cards
80

5’ end

At what end is Reporter Dye seen?

New cards
81

3’ end

At what end is Quencher seen?

New cards
82

True

Modified True or False.

In TaqMan, when the quencher and reporter dye are together, there will be no fluorescence signal.

New cards
83

Reporter Dye

This contains the fluorescence signal.

New cards
84

TaqMan signal fluorescence

It is generated when Taq polymerase extends the primers and digests the probe and releases the reporter from the vicinity of the quencher.

New cards
85

Molecular Beacon Probe

It contains target-specific sequences and a short, inverted repeat (~5 bp) that hybridizes into a hairpin structure. The 5' end of the probe has a reporter dye (R), and the 3' end has a quencher dye (Q).

New cards
86

True

Modified True or False.

In the presence of target sequences (Molecular Beacon), hybridization of the probe will open the hairpin, moving the quencher from the reporter and allowing signal fluorescence, which doubles with every doubling of target sequence.

New cards
87

Scorpion

An advantage of this system is the covalent attachment of fluorescent signal to the PCR product, which is useful for further analysis, such as size assessment by capillary electrophoresis.

New cards
88

False.

FRET probes are separate oligomers, one covalently attached to a donor fluor (D) and one to an acceptor or reporter fluor (R). The acceptor/reporter will fluoresce only when both probes are bound next to one another on the target sequences. As more target accumulates, more probes bind, and more fluorescence is emitted.

Modified True or False.

FRET probes are separate oligomers, one covalently attached to a donor fluor (D) and one to an acceptor or reporter fluor (R). The acceptor/reporter will fluoresce only when both probes are bound next to one another on the target sequences. As more target accumulates, less probes bind, and more fluorescence is emitted.

New cards
89

Sigmoidal curve

True amplification displays what curve?

New cards
90

Exponential phase

This phase refers to the increase of DNA copies.

New cards
91

Plateau phase

The enzymes increased are finished and there are no bases to be added, it has reached what phase?

New cards
92

Baseline

Low-level signals attributed to the background or the "noise" of the reaction.

New cards
93
  • Bubbles

  • Vibrations

  • Incorrect pipetting

PCR is sensitive. If there is noise, it means it is unstable. It can be caused by?

New cards
94

Treshold line

The minimum level of fluorescence to determine a CT value. It is above the baseline.

New cards
95

False.

CT is inverse to starting template amount.

Modified True or False.

CT is proportional to starting template amount.

New cards
96

CT value

When the fluorescence crosses the treshold line, it determines the?

New cards
97

Contamination

Late amplification without sigmoidal curve means?

New cards
98
  • Sigmoidal amplification

  • CT value

What are the 2 requirements to determine the result in qPCR?

New cards

Explore top notes

note Note
studied byStudied by 2 people
Updated ... ago
5.0 Stars(1)
note Note
studied byStudied by 32 people
Updated ... ago
5.0 Stars(1)
note Note
studied byStudied by 10 people
Updated ... ago
5.0 Stars(1)
note Note
studied byStudied by 2 people
Updated ... ago
5.0 Stars(1)
note Note
studied byStudied by 19 people
Updated ... ago
5.0 Stars(1)
note Note
studied byStudied by 5 people
Updated ... ago
5.0 Stars(2)
note Note
studied byStudied by 53 people
Updated ... ago
5.0 Stars(1)
note Note
studied byStudied by 248 people
Updated ... ago
5.0 Stars(1)

Explore top flashcards

flashcards Flashcard33 terms
studied byStudied by 3 people
Updated ... ago
5.0 Stars(3)
flashcards Flashcard58 terms
studied byStudied by 11 people
Updated ... ago
5.0 Stars(1)
flashcards Flashcard95 terms
studied byStudied by 9 people
Updated ... ago
5.0 Stars(1)
flashcards Flashcard92 terms
studied byStudied by 13 people
Updated ... ago
5.0 Stars(1)
flashcards Flashcard43 terms
studied byStudied by 1 person
Updated ... ago
5.0 Stars(1)
flashcards Flashcard49 terms
studied byStudied by 30 people
Updated ... ago
5.0 Stars(3)
flashcards Flashcard23 terms
studied byStudied by 50 people
Updated ... ago
5.0 Stars(4)
flashcards Flashcard89 terms
studied byStudied by 15 people
Updated ... ago
5.0 Stars(2)