PCR Flashcards
Laboratory technique to make million/billion copies of a specific region of DNA to make enough copies that can be analyzed
Polymerase chain reaction
What are the different components of PCR?
Taq DNA polymerase Primer dNTPs Buffer Template DNA/RNA Additional for RT PCR are reverse transcriptase and Probe
It is a thermostable DNA polymerase
Taq Polymerase
Taq polymerase is isolated from the bacterium ___?
Thermus aquaticus, which is a thermophilic bacteria
short sequence of nucleotides that provides a start point for DNA synthesis
Primer
provides optimal conditions for robust enzyme activity
Buffer
Buffer has a pH of __?
8.0-9.5
Extracted DNA/RNA at a suitable concentration
Template DNA/RNA
This is used to detect viral RNA and converts RNA to cDNA
Reverse Transcription- Polymerase chain reaction
Probe is made up of ___?
Fluorophore and Quencher
It is a fluorescent chemical compound that re-emits light upon light excitation
Fluorophore
It suppresses fluorescence by absorbing the light emitted by the fluorophore
Quencher
What are the steps in RT-PCR?
Reverse transcription, Denaturation, Annealing, Extension, Collect Data and Repeat Step 3-5 for 39 cycles
During this step, the enzyme converts RNA to complementary DNA
Reverse transcription
It is the part wherein the reaction components are exposed at high temperature (95C)
Denaturation
What is the purpose of denaturation?
To separate the strands of DNA, exposing its nitrogenous bases and to inactivates reverse transcriptase enzymes.
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
Annealing
In this step, no fluorescence is yet to be detected by the spectrophotometer
Annealing
In this step, Taq polymerase activates primer binding, adding complementary bases to build a new copy strand.
Extension
In this step, fluorescence is detected because the fluorophore is no longer near the proximity to the quencher.
Extension
TRUE OR FALSE:
The spectrophotometer reads the fluorescence of the bound fluorophore in the solution
FALSE
It should be the free fluorophore or the fluorescent material in the solution. The absorbance is measured providing a quantitative value
TRUE OR FALSE:
With each cycle, the number of free fluorescence material is doubled and the signal detected by the spectrophotometer should also increase
TRUE
It is done to separate the template RNA from other components of the specimen
RNA Extraction
4 steps of Nucleic acid extraction
Lysis, Binding, Washing, Elution
This step releases all contents of the cell including nucleic acids
Cell lysis
What method is used for cell lysis?
Chemical method because it disrupts the cells and do not harm the nucleic acids
RNA attachment to the filter and uses a buffer
Binding
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.
FALSE
BUFFER should have LOWER CONCENTRATION AND PH than the FILTER
The purpose of this step is for the removal of impurities and allows the contaminants to flow through the filter
Washing
-where it uses wash buffers
Addition of nuclease-free water to change the pH
Elution
TRUE OR FALSE:
Nuclease-free water has a lower salt concentration than the filter
TRUE
Mastermix preparation is done in ____?
inside of a PCR cabinet
Mixing of mastermix and template RNA
Template Addition
In this phase, you mix the mastermix with the positive template control
Template addition
In this phase, you mix the mastermix with the Negative template control
Mastermix preparation
What are the 4 phases in PCR
Baseline phase, exponential phase, linear phase and plateau phase
What genes of SARS-CoV-2 are detected in most of the PCR detection kit?
OFR1ab and N genes
Give the target specific to each dye
Cy5
FAM
ROX
Cy5-internal control
FAM-OFR1ab gene
ROX-N gene
a point when sample fluorescence rises above background fluorescence
Cycle treshold
number of cycle it takes to cross the cycle treshold
Ct value
it is a nonspecific fluorescence in the reaction
Background
QUALITY CONTROL
Negative control/No template control
NO CT or CT>40 at channel FAM, ROX, CY5
QUALITY CONTROL
Negative extraction control
NO CT or CT>40 at channel FAM, ROX, CY5
QUALITY CONTROL
Positive control
less than or equal to 35 at channel FAM, ROX, CY5
This control indicates that contamination was introduced in the PCR phase
No template control
This control indicates that contamination was introduced in the extraction phase
Negative extraction control
This control is an indicative of limit of detection and robustness of the assay
Positive template control
SARS-CoV-2 positive when
There is a typical S-Shape curve at FAM/ROX, CY5 channel CT <40
SARS-CoV-2 negative when
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.
PCR Phase includes
Mastermix and Template Addition
