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MT6319 > [LAB] Unit 4.2_The PCR Reaction Chemistry > Flashcards

[LAB] Unit 4.2_The PCR Reaction Chemistry Flashcards

1
Q

DNA denatures at what temperature?

A

90+ °C

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2
Q
  • PCR is only made possible due to the discovery of this polymerase
    -It is stable enough to withstand 90+ °C
A

Taq polymerase

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3
Q

Genus and specie of this thermostable bacteria

A

Thermus aquaticus (TAQ)

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4
Q

What are the PCR reaction components? (7)

A

-Water
-Buffer
-DNA template
-Primers
-Nucleotides
-Mg++ ions
-DNA polymerase

Do Not Play While Being MD

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5
Q

This PCR reaction component is:

-The medium for all other components
-It is specially purified, double-distilled, deionized, autoclaved, and nuclease-free

A

Water

“PCR-grade Water” yung label sa picture ng bottle

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6
Q

(T/F): We can also use Triple Distilled Water

A

True

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7
Q

This PCR reaction component:

-contains region to be amplified
-any DNA desired

A

DNA template

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8
Q

(T/F) Purity is required for DNA template

A

FALSE. Purity is NOT required.

However, there are instances wherein protein might interfere with your runs into quenching

Increase quenching = decreased in fluorescence

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9
Q

(T/F) Increase quenching = increase in fluorescence

A

FALSE.

Increase quenching = decreased in fluorescence

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10
Q

(T/F) Water should not contain detectable amounts of nucleic acids

A

True

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11
Q

This PCR reaction component:

  • stabilizes the DNA polymerase, DNA, and nucleotides
A

Buffer

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12
Q

Examples of Buffer used in PCR

A
  • 500 mM KCl
  • 100 mM Tris-HCl, ph 8.3
  • Triton X-100 or Tween
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13
Q

(T/F) In the context of Buffer, optimum pH would mean that your enzymes will work correctly

A

True

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14
Q

This PCR reaction component:

-specific ends of amplified region

-annealing temps should be known depending on its:
*length,
*GC content,
etc.
-This would tell you when to start and end your PCR

A

Primers

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15
Q

Length of Primer

A

15-30 nt

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16
Q

Concentration of Primer

A

0.1 - 1.0 uM (pMol/ul)

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17
Q

What are the two (2) types of Primers?

A

Forward and Reverse primers

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18
Q
  • This primer will tell you when to begin replicating your DNA molecule; when to begin your extension

-from 5 region to 3 region

A

Forward primer

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19
Q

-This primer tells you where to stop; where to end your extension

-nakabaliktad (3’ to 5’)

A

Reverse primer

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20
Q

(T/F) the sequence in between the forward and reverse primers are the only ones that will replicate

A

True

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21
Q

-Has a marker which helps you detect formation of the DNA segment you need

A

Probe

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22
Q

This PCR reaction component:

  • is added to the growing chain
  • Activated NTP’S
    –dATP, dGTP, dCTP, dTTP
A

Nucleotides

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23
Q

Storage condition of Nucleotides

A

Stored at 10mM, ph 7.0

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24
Q

How much Nucleotides is added in assay?

A

20-200 uM

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25
Q

These are the building blocks

A

Nucleotides

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26
Q

Nucleotides is in in the form of ___________

A

dinucleotide triphosphate (dNTPs)

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27
Q

4 types of dNTPs

A

Adenine
Guanine
Cytosine
Thymine

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28
Q

This PCR reaction component:

-is an Essential co-factor of DNA Polymerase

A

Magnesium

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29
Q

This PCR reaction component:

-the reason why EDTA is used

A

Magnesium

note: EDTA creates ions like calcium and magnesium from the blood sample via chelation

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30
Q

This PCR reaction component:

  • Stabilizes the DNA double-helix
A

Magnesium

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31
Q

Too little of Mg++ ions:

a. Enzymes won’t work
b. non-specific priming
c. Band smearing

A

a. Enzymes won’t work

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32
Q

Too much Mg++ ions:

a. will make DNA extra stable
b. Non-specific priming
c. Band smearing
d. All of the above

A

d. All of the above

a. will make DNA extra stable
b. Non-specific priming
c. Band smearing

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33
Q

Concentration of Magnesium used in the assay

A

0.5 - 3.5 uM

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34
Q

This PCR reaction component:

  • the enzyme that does the extension
A

DNA polymerase

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35
Q

This PCR reaction component:

  • TAQ or similar
  • Heat stable
A

DNA polymerase

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36
Q

(T/F) In mixing common reagents, add additional 1-2x over and above the required volume

A

True.

To compensate for pipetting errors

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37
Q

What is next after the last step in mixing common reagents?

A

Adding the DNA

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38
Q

This happens when the DNA molecules splits into two

A

Denaturation

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39
Q

This happens when the primer starts binding to the DNA

A

Annealing

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40
Q

This device gives an environment wherein your sample can encounter temperatures that allow for denaturing, annealing, and extension

A

Thermal cycler

41
Q

This happens when the DNA polymerase starts adding nucleotide bases

A

Extension

42
Q

(T/F) The copy of your DNA products are independent on the number of cycles

A

False.

Dependent

43
Q

How to estimate the number of molecules in a PCR run?

A

2ⁿ

2 = constant
and raise it by the power of n
n = number of cycles

44
Q

You subjected your PCR run in 30 cycles.

what is your estimate number of copies?

A

at least 2³⁰ copies

45
Q

(T/F)
The more you subject your enzymes to cycling temperatures, the less effective they become.

A

True

46
Q

(T/F)
If you subject your Taq polymerase enzyme to repeated temperature variations, its effectivity would not be affected.

A

False.

Like most things, heat will eventually degrade things over time.

If you subject your Taq polymerase enzyme to repeated temperature variations, mawawala yung effectivity ng enzymes.

47
Q

(T/F)
PCR run is dependent on the amount of dnTPs that you put in

A

True.

You can’t build a skyscraper with building blocks only enough for a bungalow

48
Q

Visualizing results:

After thermal cycling, tubes are taken out of the PCR machine. The contents of the tubes are loaded onto an _______ ___

A

Agarose gel

49
Q

Visualizing results:

PCR products are visible as different “_____s”

A

Bands

50
Q

What is the end product of a PCR run?

A

Amplicon

51
Q

After a cycle run, what is left in your reaction mix is your _______

A

Amplicon

52
Q

Modification to Traditional PCR
(3)

A

Nested PCR
Multiplex PCR
Digital PCR

53
Q

This PCR occurs when you amplify a large region of your DNA molecule, then once you are able to replicate that segment, you will do another PCR of a smaller section of that fragment.

A

Nested PCR

53
Q

What is the major concern for Nested PCR?

A

contamination that occurs during the transfer of the first-round product to the second tube for the second round of amplification

54
Q

This PCR was developed to increase both the sensitivity and specificity of PCR

A

Nested PCR

55
Q

This technique uses two pairs of amplification primers and two rounds of PCR

A

Nested PCR

56
Q

In this PCR, multiple primer sets designed for amplification of different targets are included in the same PCR reaction

(2 or more primer sets used in one PCR run)

A

Multiplex PCR

56
Q

Example of usage for Nested PCR

A

Determining the hair color

57
Q

What is the prevention step for the major concern for Nested PCR?

A

by physically separating the first- and second-round amplification mixtures with a layer of wax oil.

58
Q

Where is Nested PCR used?
a. genome polymorphisms
b. cloning
c. methylation analysis

A

a. Genome Polymorphisms

-Insertions
-Deletions
-SNPs

59
Q

Example of use of Multiplex PCR

A

PCR for HIV

If there are 3 HIV-related genes that we want to test from the patient, just load 1 PCR run with 3 primer sets

60
Q

(T/F) Multiplex PCR is practical and can’t not save time and effort

A

True

it can’t not = it can

61
Q

(T/F) In Multiplex PCR, we can use a set of primers as internal control so that we can eliminate the possibility of false positives or negative

A

True

61
Q

(T/F)
In multiplex PCR, the amplicon sizes of your different primer sets should be DIFFERENT ENOUGH in terms of the number of base pairs / molecular weight so that they can be visualized properly using gel electrophoresis.

A

True

61
Q

Although Multiplex PCR can reduce costs and time to simultaneously detect several pathogens in a specimen, it is often _______ than single-primer-set PCR

A

less sensitive

62
Q

(T/F)
Multiplex PCR can only be designed for a single-template PCR reaction containing several sets of primers

A

False.

Multiplex PCR can be designed in either single-template or multiple-template PCR reaction,

63
Q

Multiplex PCR is more complicated due to the possible interaction of different primers that has _____-_____ possibilities

A

primer-dimer

64
Q

This PCR can save costly polymerase and template in short supply.

A

Multiplex PCR

65
Q

This PCR increases the accuracy of the molecular PCR because it transforms the exponential data to digital signals

A

Digital PCR

66
Q

(T/F)
Digital PCR can be applied if we want to detect and quantify low levels of pathogen sequences

A

True

66
Q

This PCR allows for quantification of individual DNA molecules

A

Digital PCR

66
Q

The Digital PCR computation is dependent on the ______ distribution

A

Poisson distribution

67
Q

(T/F)
Digital PCR can be applied if we want to detect and quantify the expression of rare genetic sequences in single cells

A

True

68
Q

(T/F)
Digital PCR can be applied if we want to detect and quantify the clonal amplification of nucleic acids for sequencing mixed nucleic acid samples.

A

True

69
Q

This test is done to make sure if the amplicon is correctly done by way of producing cleavage if a restriction enzyme is used

A

Restriction enzyme cleavage

69
Q

Digital PCR is ideal for _______

A

Cloning

70
Q

This test is used to check if the base pairs are amplified correctly

A

Gel electrophoresis

70
Q

Tests to Identify the Quality of PCR product

A
  • Gel electrophoresis
  • Capillary electrophoresis
  • Restriction enzyme cleavage
  • Nucleic acid hybridization
  • DNA sequencing
  • Real-time quantitative PCR

Cutter GRNDR

70
Q

This test has the same principle as gel electrophoresis, however uses a different medium (capillary sieving gel)

A

Capillary electrophoresis

71
Q

This test can be combined with a probe or a molecule that detects a specific sequence in the amplicon

A

Nucleic acid hybridization

71
Q

What test is this?

An enzyme only reacts to a particular amplicon or gene sequence, if it cleaves = correct PCR reaction performed

A

Restriction enzyme cleavage

72
Q

In this test, if an amplification is found = DNA is copied or amplified

A

Real-time quantitative PCR

72
Q

This test is performed in a machine to check if the applied DNA base pairs are correct

A

DNA sequencing

73
Q

This PCR is more complex because in PCR we use fluorometry for measuring PCR output.

A

Quantitative PCR / qPCR

73
Q

In performing conventional PCR, what is usually expected is the amplified DNA or the _______.

So in order to quantify how much DNA is produced, we must use this special type of PCR called _ _ _ _.

A

amplicon

qPCR

74
Q

In qPCR, aside from the forward and reverse primer, there is another sequence, which is the _____.

A

Probe

75
Q

This DNA sequence is connected to a fluorescent marker, and are expected to fit within the sequences produced by the forward and reverse primers.

A

Probe

75
Q

(T/F)

DNA is used to detect the presence of a gene

RNA is used to detect the level of expression of the gene

A

True

76
Q

Where and when should we use PCR?:

To know the _______ of a ________

Ex.
To identify the presence of hepatitis B virus, we must look for a gene that is only specific to Hepa B virus.

A

presence of a gene expression

77
Q

(T/F)
In order to perform RNA PCR, reverse transcription should be performed to make DNA from RNA.

The product will be the formation of complementary DNA (cDNA) from the RNA molecule.

cDNA is now used for PCR.

A

True

78
Q

Top Applications for PCR

A
  • Gene expressoin
  • Genotyping
  • Cloning
  • Mutagenesis
  • Methylation Analysos
  • Sequencing
  • Medical, forensics, and applied sciences
79
Q

+/+
-This is the normal gene
- Here, both strands of your DNA are normal

A

Wildtype +/+

80
Q

In RT-PCR, _______ _______ will bind DNA to your RNA template.

After it reaches your (Answer 1), you now have a ___-___ ______

Summary:
From an RNA molecule, we subjected it to reverse transcription, forming a complementary DNA, and then we can use that for PCR.

A

reverse transcriptase

DNA-RNA hybrid

81
Q

+/-
-This means that of the copies do not match the wild type

A

Heterozygous +/-

81
Q

This is a method that allows you to look into the differences in the nucleotide bases in each allele

It can detect mismatched nucleotide bases (Ex. when adenine binds with cytosine)

A

Allelic Discrimination

82
Q

-/-
-If you have both copies that do not match the wild type

A

Homozygous -/-

83
Q

Different types of Modern PCR Method

A

Direct
Indirect

84
Q

Future of PCR

A
  • Copying larger pieces of DNA
  • Miniaturization of hardware (chip-sized)
  • Computer automated test and analysis
  • Mobile testing
  • Rapid turnaround time

MT6319 (4 decks)

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