10.6 Mechanism of Pcr Reaction

PCR cycle consists of three steps: denaturation, primer annealing and extension or polymerization. Each step requires a specific temperature. The time duration, temperature and sequence of the steps have to be programmed in the thermocycler.

Definition

Mechanism of PCR Reaction: PCR cycle consists of three steps: denaturation, primer annealing and extension or polymerization. Each step requires a specific temperature. The time duration, temperature and sequence of the steps have to be programmed in the thermocycler.

Main Idea

• PCR cycle consists of three steps:
• Each step requires a specific temperature.
• The time duration, temperature and sequence of the steps have to be programmed in the thermocycler.
• Denaturation has two types:
• Initial denaturation — performed only once before cyclic steps
• Cyclic denaturation — performed in each cycle
• Extension has two types:
• Cyclic extension — performed in each cycle
• Final extension — performed only once after cyclic steps
• At the end of first cycle, one target dsDNA molecule is converted into two daughter dsDNA molecules.
• The second cycle immediately starts with denaturation.
• The cycle continues resulting in amplification at an exponential rate.
• The number of existing DNA molecules becomes doubled after each cycle.
• Initial denaturation and final extension contribute to the efficiency and accuracy of PCR.
• Storage: At the end of the extension, temperature is set at 4°C for longer time.

Core Concept

• PCR cycle = denaturation, primer annealing, extension
• Initial denaturation = once; 94-95°C for 4-5 minutes
• Cyclic denaturation = each cycle; 94°C for 1 minute
• Primer annealing = each cycle; 55-65°C for 2 minutes
• Cyclic extension = each cycle; 72°C for 1-2 minutes
• Final extension = once; 72°C for 5-7 minutes
• **Storage** = 4°C
• Amplification = exponential rate
• Number of DNA molecules = doubled after each cycle

Detailed Explanation

Mechanism of PCR Reaction

PCR cycle consists of three steps: denaturation, primer annealing and extension or polymerization. Each step requires a specific temperature. The time duration, temperature and sequence of the steps have to be programmed in the thermocycler.

Denaturation

Initial Denaturation

• This step is performed only once before the start of cyclic steps of PCR process.
• Purpose: To completely denature the DNA template and ensure all strands are separated before cycling begins.
• Temperature and time (typically) set at 94-95°C for 4-5 minutes.
• Reason: Some DNA templates, especially GC-rich regions or complex secondary structures, require a longer initial denaturation to break hydrogen bonds fully, allowing proper primer binding.

Cyclic Denaturation

• Cyclic denaturation step is performed in each cycle of the PCR process.
• The template DNA is heated to 94°C for one minute.
• At this high temperature the DNA undergoes complete denaturation and the double-stranded DNA (dsDNA) becomes single-stranded DNA (ssDNA).
• Each single ssDNA can act as the template for the in vitro DNA synthesis.

Primer Annealing

• The next step is the primer annealing.
• In this step the two primers, the forward primers and the reverse primers, anneal or hybridize to the single-stranded template DNA at its complementary regions.
• Annealing is usually carried out at a lower temperature depending on the length and sequence of the primers.
• In standard cases temperature is between 55°-65°C depending upon size of the primer and the length of the gene.
• Approximate time required for this step is 2 minutes.

Extension or Polymerization

Cyclic Extension

• Cyclic Extension is performed in each cycle of the PCR process.
• This is final step in each cycle of primer extension or polymerization in which the Taq polymerase synthesizes new DNA strands to the 3' ends of primers using dNTPs.
• The optimum temperature for carrying out the primer extension reaction or polymerization of dNTPs is standardized at 72°C.
• This step takes just one minute to be completed in each cycle.

Final Extension

• On the completion of cyclic steps of PCR process final extension is performed only once.
• Purpose: For complete synthesis of all PCR products by Taq DNA polymerase.
• Temperature and time is usually 72°C for 5-7 minutes.
• Reason: The final extension ensures that any incomplete remaining DNA strands during cyclic extension, are fully extended resulting in complete double-stranded products.

Amplification

• At the end of first cycle, one target dsDNA molecule is converted into two daughter dsDNA molecules.
• The second cycle immediately starts with the denaturation by heating at 94°C, so that all the newly synthesized dsDNA molecules are also denatured to single strands, which again act as templates.
• It will again be followed by the primer annealing and extension and thus the cycle of denaturation, primer annealing, and extension continues resulting in the amplification of the selected DNA sequence at an exponential rate i.e., the number of existing DNA molecules become doubled after each cycle.
• Both non cyclic steps i.e. initial denaturation and final extension contribute to the efficiency and accuracy of PCR, leading to better yields and more reliable results.

Storage

- At the end of the extension, temperature is set at 4°C for longer time to safely store the PCR products (i.e. replicated DNA copies) until it is not shifted to refrigerator.

Summary Table of PCR Steps

Summary of Amplification

Intermediate Level

PCR Steps

Amplification

Purpose of Non-Cyclic Steps

Formula / Mathematical Form

Amplification Formula: Number of DNA molecules = 2ⁿ

Where: - n = number of cycles

Example: If n = 10: 2¹⁰ = 1024 molecules

Solved Example

**Given:** Number of cycles = 10

Required: Number of DNA molecules

Formula: Number of DNA molecules = 2ⁿ

Substitution: 2¹⁰

Calculation: 2¹⁰ = 1024

Final Answer: After 10 cycles, there will be 1024 DNA molecules.

Important Points

• 1. PCR cycle has three steps: denaturation, primer annealing, extension.
• 2. Each step requires a specific temperature.
• 3. Initial denaturation is performed once.
• 4. Initial denaturation is at 94-95°C for 4-5 minutes.
• 5. Cyclic denaturation is performed in each cycle.
• 6. Cyclic denaturation is at 94°C for 1 minute.
• 7. Primer annealing is at 55-65°C for 2 minutes.
• 8. Cyclic extension is at 72°C for 1-2 minutes.
• 9. Final extension is performed once.
• 10. Final extension is at 72°C for 5-7 minutes.
• 11. Storage is at 4°C.
• 12. At the end of first cycle, one dsDNA becomes two dsDNA.
• 13. Second cycle starts with denaturation.
• 14. Amplification occurs at exponential rate.
• 15. Number of DNA molecules doubles after each cycle.
• 16. Initial denaturation and final extension contribute to efficiency.
• 17. They lead to better yields and more reliable results.
• 18. PCR products are stored at 4°C.
• 19. PCR is used in research, medicine, and forensic science.
• 20. PCR is essential for genetic engineering.

Remember

• - PCR cycle = denaturation, primer annealing, extension
• - Initial denaturation = once; 94-95°C for 4-5 minutes
• - Cyclic denaturation = each cycle; 94°C for 1 minute
• - Primer annealing = each cycle; 55-65°C for 2 minutes
• - Cyclic extension = each cycle; 72°C for 1-2 minutes
• - Final extension = once; 72°C for 5-7 minutes
• - **Storage** = 4°C
• - Amplification = exponential rate
• - Number of DNA molecules = doubled after each cycle
• - **Formula** = 2ⁿ

Common Mistakes

• 1. Confusing initial and cyclic denaturation — Initial = once; cyclic = each cycle.
• 2. Mixing up the temperatures — Denaturation: 94°C; annealing: 55-65°C; extension: 72°C.
• 3. Forgetting the role of final extension — Complete synthesis of all PCR products.
• 4. Mixing up the duration of steps — Initial denaturation: 4-5 min; cyclic denaturation: 1 min; annealing: 2 min; extension: 1-2 min; final extension: 5-7 min.
• 5. Thinking amplification is linear — Amplification is exponential.
• 6. Forgetting the formula — Number of DNA molecules = 2ⁿ.
• 7. Mixing up the storage temperature — 4°C.
• 8. Believing PCR is a single step — PCR has multiple steps.
• 9. Mixing up the purpose of non-cyclic steps — Initial denaturation and final extension contribute to efficiency.
• 10. Thinking PCR is not important — PCR is a fundamental technique in molecular biology.

Quick Revision

PCR cycle consists of three steps: denaturation, primer annealing and extension or polymerization. Each step requires a specific temperature. The time duration, temperature and sequence of the steps have to be programmed in the thermocycler. Initial Denaturation: This step is performed only once before the start of cyclic steps of PCR process. Purpose: To completely denature the DNA template and ensure all strands are separated before cycling begins. Temperature and time (typically) set at 94-95°C for 4-5 minutes. Reason: Some DNA templates, especially GC-rich regions or complex secondary structures, require a longer initial denaturation to break hydrogen bonds fully, allowing proper primer binding. Cyclic Denaturation: Cyclic denaturation step is performed in each cycle of the PCR process. The template DNA is heated to 94°C for one minute. At this high temperature the DNA undergoes complete denaturation and the double-stranded DNA (dsDNA) becomes single-stranded DNA (ssDNA). Each single ssDNA can act as the template for the in vitro DNA synthesis. Primer annealing: The next step is the primer annealing. In this step the two primers, the forward primers and the reverse primers, anneal or hybridize to the single-stranded template DNA at its complementary regions. Annealing is usually carried out at a lower temperature depending on the length and sequence of the primers. In standard cases temperature is between 55°-65°C depending upon size of the primer and the length of the gene. Approximate time required for this step is 2 minutes. Extension or Polymerization: Cyclic Extension: Cyclic Extension is performed in each cycle of the PCR process. This is final step in each cycle of primer extension or polymerization in which the Taq polymerase synthesizes new DNA strands to the 3' ends of primers using dNTPs. The optimum temperature for carrying out the primer extension reaction or polymerization of dNTPs is standardized at 72°C. This step takes just one minute to be completed in each cycle. Final Extension: On the completion of cyclic steps of PCR process final extension is performed only once. Purpose: For complete synthesis of all PCR products by Taq DNA polymerase. Temperature and time is usually 72°C for 5-7 minutes. Reason: The final extension ensures that any incomplete remaining DNA strands during cyclic extension, are fully extended resulting in complete double-stranded products. At the end of first cycle, one target dsDNA molecule is converted into two daughter dsDNA molecules. The second cycle immediately starts with the denaturation by heating at 94°C, so that all the newly synthesized dsDNA molecules are also denatured to single strands, which again act as templates. It will again be followed by the primer annealing and extension and thus the cycle of denaturation, primer annealing, and extension continues resulting in the amplification of the selected DNA sequence at an exponential rate i.e., the number of existing DNA molecules become doubled after each cycle. Both non cyclic steps i.e. initial denaturation and final extension contribute to the efficiency and accuracy of PCR, leading to better yields and more reliable results. Storage: At the end of the extension, temperature is set at 4°C for longer time to safely store the PCR products (i.e. replicated DNA copies) until it is not shifted to refrigerator.

60-Second Revision

• PCR cycle = denaturation, primer annealing, extension
• Initial denaturation = once; 94-95°C for 4-5 minutes
• Cyclic denaturation = each cycle; 94°C for 1 minute
• Primer annealing = each cycle; 55-65°C for 2 minutes
• Cyclic extension = each cycle; 72°C for 1-2 minutes
• Final extension = once; 72°C for 5-7 minutes
• Storage = 4°C
• Amplification = exponential rate
• Number of DNA molecules = doubled after each cycle
• Formula = 2ⁿ
• Warning: Amplification is exponential, not linear

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