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Questions and Answers
What is the main purpose of the Polymerase Chain Reaction (PCR)?
What is the main purpose of the Polymerase Chain Reaction (PCR)?
Which component is NOT required for the PCR process?
Which component is NOT required for the PCR process?
During which stage of PCR do the DNA strands separate?
During which stage of PCR do the DNA strands separate?
What is the temperature range used during the annealing step of PCR?
What is the temperature range used during the annealing step of PCR?
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What role do primers play in the PCR process?
What role do primers play in the PCR process?
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What is the purpose of DNA polymerase during the extension step?
What is the purpose of DNA polymerase during the extension step?
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At which temperature do the primers anneal to the DNA strands?
At which temperature do the primers anneal to the DNA strands?
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What is the primary function of the initial melt step at 94ºC?
What is the primary function of the initial melt step at 94ºC?
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Which step follows the annealing of primers during the DNA replication process?
Which step follows the annealing of primers during the DNA replication process?
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What occurs during the final extension phase at 72ºC?
What occurs during the final extension phase at 72ºC?
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Study Notes
Polymerase Chain Reaction (PCR)
- PCR is an in-vitro technique used to amplify a specific DNA region.
- This technique is used to create numerous copies of a DNA segment.
- Using PCR, billions of copies of a target DNA sequence can be made in a few hours.
- The target DNA sequence can be known or located between two known sequences.
Reaction Components
-
Reagents Needed:
- DNA sample to be amplified
- DNA polymerase (Taq polymerase—works at high temps up to 95°C)
- Nucleotides (dNTPs)
- Primers (one primer to the 5' end of a DNA strand, the other to the 3' end of the anti-parallel strand)
- A suitable apparatus to perform 35 cycles of a 3-temperature process: 95°C, 50-60°C, and 72°C.
PCR Thermal Cycler
- The DNA sample, DNA polymerase, buffer, nucleotides, and primers are placed in a thin-walled tube.
- These tubes are then placed in the PCR thermal cycler.
PCR Protocol
- Melting/Heat Denaturation: The DNA double strand breaks down into two single strands (5' to 3' and 3' to 5').
- Annealing: Primers bind to their complementary sequences on single strands. (5' primer to 5' to 3' strand; 3' primer to 3' to 5' strand)
- Extending: DNA polymerase copies each strand by adding nucleotides to the 3' ends of the primers, creating new DNA strands.
Temperature Protocol
- Initial Melt: 94°C for 10 minutes
- Melt: 95°C for 30 seconds
- Anneal: 55°C for 30 seconds
- Extend: 72°C for 1 minute
- Final Extension: 72°C for 6 minutes
- Hold: 4°C
Steps of PCR (Detailed)
- Step 1: Denature DNA: At 95°C, the DNA is denatured, separating the two strands.
- Step 2: Primers Anneal: Temperature is lowered (Tm) and primers anneal to target sequences.
- Step 3: DNA Polymerase Extends: At 72°C, DNA polymerase extends the DNA chain by adding nucleotides to the 3' ends of the primers.
Gel Electrophoresis of DNA
- Gel electrophoresis detects the presence of DNA, and the number of nucleotides in a DNA fragment.
- This technique is useful in measuring the DNA regions amplified using PCR.
- Fragments of different lengths move at different speeds through the gel.
- The results are visualized with a special dye, and fragments are compared to a known sample to estimate the number of nucleotides.
PCR Types & Variations
- Multiplex PCR
- Asymmetric PCR
- Hot-start PCR
- Inverse PCR
- Nested PCR
- Quantitative PCR (Q-PCR)
- Reverse Transcription PCR (RT-PCR)
- Thermal asymmetric interlaced PCR (TAIL-PCR)
- Touchdown PCR (Step-down PCR)
Advantages of PCR
- Extremely high sensitivity—can detect a single viral genome per sample.
- Easy to set up
- Fast turnaround time
Disadvantages of PCR
- Extremely susceptible to contamination.
- High degree of operator skill required
- Difficulty in interpreting a positive result, especially with latent viruses (like CMV).
Medical Applications of PCR
- Amplification and quantification of DNA and RNA.
- Diagnosis of diseases.
- PCR for comparing different genomes.
- Drug therapy
- Genetic disorders diagnosis (like thalassemia and CML)
- Pathogenic microorganism detection (viruses, bacteria, parasites, fungi)
- Gene therapy monitoring
- Molecular diagnostics and biochemical analyses
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Description
Test your knowledge on the Polymerase Chain Reaction (PCR) process with this quiz. Answer questions related to the components, stages, and temperatures involved in PCR. Perfect for students studying molecular biology and genetics.
