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Questions and Answers
What is the primary purpose of using fluorescent dyes in real-time PCR?
Which method does not involve the use of DNA probes in real-time PCR?
What is the significance of the threshold cycle (CT) in real-time PCR?
How does the 5’-3’ exonuclease activity of Taq polymerase contribute to real-time PCR detection?
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What observation can be made from the sigmoid curve of a real-time PCR plot?
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What is a significant limitation of multiplex PCR?
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What is the primary purpose of reverse transcriptase in RT-PCR?
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Why is mRNA not directly amplified by PCR?
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Which step is NOT part of the RT-PCR process?
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What is a benefit of nested PCR in clinical applications?
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What is the function of an oligo(T) primer in RT-PCR?
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Which characteristic makes reverse transcriptase unique?
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What occurs to the RNA-cDNA hybrid during the RT-PCR process?
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Study Notes
Types of PCR
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Multiplex PCR
- Allows use of multiple primer pairs in a single PCR reaction.
- Enables simultaneous amplification of various products, useful for typing and identification analyses.
- Requires complex optimization of reagents and conditions to ensure efficiency.
- Target sequences may amplify at different rates, and primer competition can occur.
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Reverse Transcriptase PCR (RT-PCR)
- Utilized for measuring gene expression by converting RNA into complementary DNA (cDNA).
- cDNA synthesis involves reverse transcriptase, which catalyzes the conversion of RNA templates into DNA.
- Retroviruses, which contain RNA genomes, utilize reverse transcriptase to create cDNA for host compatibility.
- Key steps in RT-PCR:
- Oligo(T) primers bind to the poly(A) tail of mRNA.
- Reverse transcriptase synthesizes cDNA strands.
- The RNA-cDNA hybrid is separated using increased temperature.
- Gene-specific primers anneal to complementary sequences, allowing amplification.
- DNA polymerase creates new DNA strands; the cycle is repeated to amplify the target gene, resulting in billions of copies.
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Nested PCR
- Provides enhanced sensitivity suitable for clinical applications, especially beneficial for low-quality or limited quantity samples.
- Addresses challenges with regular PCR reliability when dealing with low target levels and interfering sequences.
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Real-Time (Quantitative) PCR (qPCR)
- Measures PCR product accumulation in real-time after each amplification cycle.
- Fluorescent dyes (e.g., SYBR Green, EtBr) are utilized to label products during amplification.
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Detection Methods:
- DNA intercalating or minor groove-targeting dyes increase fluorescence upon binding to DNA.
- Fluorophore-labeled probes bind to specific sequences; Taq polymerase's exonuclease activity separates the fluorophore from the quencher, enhancing signal.
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Threshold Cycle (CT)
- Refers to the PCR cycle where sample fluorescence exceeds a defined threshold.
- A plot of PCR product accumulation typically resembles a sigmoid curve; more initial template leads to earlier fluorescence detection.
- The cycle number at which fluorescence crosses the threshold is inversely proportional to the initial amount of template.
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