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
