[27] Real-Time PCR (qPCR) Flashcards
What is Real-Time PCR (qPCR)?
A laboratory method used for the detection and quantification of a specific DNA sequence in a sample.
What does qPCR stand for?
Quantitative Polymerase Chain Reaction.
What makes qPCR different from standard PCR?
qPCR allows the detection of a DNA sequence during the PCR (Real-Time), not at the end, which provides quantitative data.
What are the main steps in a qPCR process?
- DNA template preparation
- Primer design
- PCR amplification with monitoring
- Data analysis
What is a DNA template in qPCR?
The original DNA sequence that will be copied in the PCR process.
What are primers in the context of qPCR?
Short pieces of DNA that are complementary to the DNA sequence to be amplified.
How does the PCR amplification process work in qPCR?
It works through a cycle of heating and cooling where the DNA template is copied. The copying is monitored in real-time.
What is the purpose of data analysis in qPCR?
To determine the presence and quantity of the DNA sequence of interest.
What are the common uses of qPCR?
- Gene expression analysis
- Genotyping
- Genetic variation analysis
- Pathogen detection
What is gene expression analysis in the context of qPCR?
The use of qPCR to measure the expression level of a specific gene.
What is genotyping in the context of qPCR?
The use of qPCR to determine the genetic makeup or variant of an organism.
What is genetic variation analysis in the context of qPCR?
The use of qPCR to detect and measure variations in the DNA sequence.
How is qPCR used for pathogen detection?
qPCR can identify and quantify the DNA of pathogens, allowing for rapid and accurate diagnosis.
How does real-time monitoring in qPCR work?
Fluorescent markers are included in the reaction. As the DNA sequence is amplified, the fluorescence increases and is measured at each PCR cycle.
What is a PCR cycle in qPCR?
A single round of DNA denaturation (separation), annealing (primer binding), and extension (DNA synthesis).
What is denaturation in the context of qPCR?
The separation of the two strands of the DNA molecule.
What is annealing in the context of qPCR?
The binding of primers to the single-stranded DNA.
What is extension in the context of qPCR?
The synthesis of new DNA strands using the primers as a starting point.
How is the quantity of a DNA sequence determined in qPCR?
By the cycle number when the fluorescence signal crosses a certain threshold. This is known as the cycle threshold (Ct).
What is Ct in the context of qPCR?
Cycle Threshold, the PCR cycle number at which the fluorescence signal crosses a predefined threshold. It is inversely proportional to the amount of target DNA in the sample.
What is PCR?
Polymerase Chain Reaction, a method widely used in molecular biology to make many copies of a specific DNA segment.
What is DNA?
Deoxyribonucleic acid, a molecule composed of two chains that coil around each other to form a double helix carrying genetic instructions.
