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Molec Lab > PCR > Flashcards

PCR Flashcards

1
Q

Laboratory technique to make million/billion copies of a specific region of DNA to make enough copies that can be analyzed

A

Polymerase chain reaction

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2
Q

What are the different components of PCR?

A 
Taq DNA polymerase
Primer
dNTPs
Buffer
Template DNA/RNA
Additional for RT PCR are reverse transcriptase and Probe
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3
Q

It is a thermostable DNA polymerase

A

Taq Polymerase

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4
Q

Taq polymerase is isolated from the bacterium ___?

A

Thermus aquaticus, which is a thermophilic bacteria

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5
Q

short sequence of nucleotides that provides a start point for DNA synthesis

A

Primer

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6
Q

provides optimal conditions for robust enzyme activity

A

Buffer

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7
Q

Buffer has a pH of __?

A

8.0-9.5

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8
Q

Extracted DNA/RNA at a suitable concentration

A

Template DNA/RNA

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9
Q

This is used to detect viral RNA and converts RNA to cDNA

A

Reverse Transcription- Polymerase chain reaction

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10
Q

Probe is made up of ___?

A

Fluorophore and Quencher

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11
Q

It is a fluorescent chemical compound that re-emits light upon light excitation

A

Fluorophore

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12
Q

It suppresses fluorescence by absorbing the light emitted by the fluorophore

A

Quencher

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13
Q

What are the steps in RT-PCR?

A

Reverse transcription, Denaturation, Annealing, Extension, Collect Data and Repeat Step 3-5 for 39 cycles

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14
Q

During this step, the enzyme converts RNA to complementary DNA

A

Reverse transcription

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15
Q

It is the part wherein the reaction components are exposed at high temperature (95C)

A

Denaturation

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16
Q

What is the purpose of denaturation?

A

To separate the strands of DNA, exposing its nitrogenous bases and to inactivates reverse transcriptase enzymes.

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17
Q

At this step, the temperature cools down so that the primer and the probe can bind to the complementary sequence on the single-stranded DNA template

A

Annealing

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18
Q

In this step, no fluorescence is yet to be detected by the spectrophotometer

A

Annealing

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19
Q

In this step, Taq polymerase activates primer binding, adding complementary bases to build a new copy strand.

A

Extension

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20
Q

In this step, fluorescence is detected because the fluorophore is no longer near the proximity to the quencher.

A

Extension

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21
Q

TRUE OR FALSE:

The spectrophotometer reads the fluorescence of the bound fluorophore in the solution

A

FALSE
It should be the free fluorophore or the fluorescent material in the solution. The absorbance is measured providing a quantitative value

22
Q

TRUE OR FALSE:
With each cycle, the number of free fluorescence material is doubled and the signal detected by the spectrophotometer should also increase

A

TRUE

23
Q

It is done to separate the template RNA from other components of the specimen

A

RNA Extraction

24
Q

4 steps of Nucleic acid extraction

A

Lysis, Binding, Washing, Elution

25
Q

This step releases all contents of the cell including nucleic acids

A

Cell lysis

26
Q

What method is used for cell lysis?

A

Chemical method because it disrupts the cells and do not harm the nucleic acids

27
Q

RNA attachment to the filter and uses a buffer

A

Binding

28
Q

TRUE OR FALSE:
Precipitate nucleic acids binds to the silica and filter of spring column when the FILTER has a lower pH and lower concentration than the BUFFER.

A

FALSE

BUFFER should have LOWER CONCENTRATION AND PH than the FILTER

29
Q

The purpose of this step is for the removal of impurities and allows the contaminants to flow through the filter

A

Washing

-where it uses wash buffers

30
Q

Addition of nuclease-free water to change the pH

A

Elution

31
Q

TRUE OR FALSE:

Nuclease-free water has a lower salt concentration than the filter

A

TRUE

32
Q

Mastermix preparation is done in ____?

A

inside of a PCR cabinet

33
Q

Mixing of mastermix and template RNA

A

Template Addition

34
Q

In this phase, you mix the mastermix with the positive template control

A

Template addition

35
Q

In this phase, you mix the mastermix with the Negative template control

A

Mastermix preparation

36
Q

What are the 4 phases in PCR

A

Baseline phase, exponential phase, linear phase and plateau phase

37
Q

What genes of SARS-CoV-2 are detected in most of the PCR detection kit?

A

OFR1ab and N genes

38
Q

Give the target specific to each dye
Cy5
FAM
ROX

A

Cy5-internal control
FAM-OFR1ab gene
ROX-N gene

39
Q

a point when sample fluorescence rises above background fluorescence

A

Cycle treshold

40
Q

number of cycle it takes to cross the cycle treshold

A

Ct value

41
Q

it is a nonspecific fluorescence in the reaction

A

Background

42
Q

QUALITY CONTROL

Negative control/No template control

A

NO CT or CT>40 at channel FAM, ROX, CY5

43
Q

QUALITY CONTROL

Negative extraction control

A

NO CT or CT>40 at channel FAM, ROX, CY5

44
Q

QUALITY CONTROL

Positive control

A

less than or equal to 35 at channel FAM, ROX, CY5

45
Q

This control indicates that contamination was introduced in the PCR phase

A

No template control

46
Q

This control indicates that contamination was introduced in the extraction phase

A

Negative extraction control

47
Q

This control is an indicative of limit of detection and robustness of the assay

A

Positive template control

48
Q

SARS-CoV-2 positive when

A

There is a typical S-Shape curve at FAM/ROX, CY5 channel CT <40

49
Q

SARS-CoV-2 negative when

A

No typical S-Shape curve at FAM/ROX (NO CT) or CT>40 detected at FAM/ROX, and CY5 less than or equal to 40.

50
Q

PCR Phase includes

A

Mastermix and Template Addition

Molec Lab (6 decks)

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