10.5 Polymerase Chain Reaction (pcr)

The polymerase chain reaction (PCR) is a technique used to amplify (clone) a single gene or a piece of DNA into thousands to millions of copies by means of an in-vitro replication process. It was invented by Kary B. Mullis in 1983. PCR uses a special DNA polymerase, Taq polymerase, which is temperature-tolerant and stable at near-boiling temperatures.

Definition

Polymerase Chain Reaction (PCR): The technique, which is used to amplify (clone) a single gene or a piece of DNA into thousands to millions of copies by means of in-vitro replication process is called polymerase chain reaction (PCR).

Main Idea

• PCR is a technique used to amplify a single gene or a piece of DNA.
• It produces thousands to millions of copies.
• It is an in-vitro replication process.
• DNA polymerase is compelled to polymerize a given piece of DNA again and again.
• This produces multiple copies.
• PCR was invented by Kary B. Mullis in 1983.
• He was awarded the Nobel Prize in Chemistry in 1993.
• A special DNA polymerase, Taq polymerase, is used.
• Taq polymerase is a temperature-tolerant enzyme.
• It is isolated from Thermus aquaticus, a bacterium found in hot springs.
• This enzyme is stable and active at near-boiling temperatures.
• For PCR, the following are needed:
• Template DNA (DNA to be amplified)
• Free nucleotides (dNTPs)
• Primers
• Taq polymerase
• These are diluted in a suitable buffer to make the PCR reaction mixture.
• The PCR mixture is placed in an instrument called thermocycler or PCR machine.
• Thermocycler regulates the temperature during various steps of PCR reaction.

Core Concept

• PCR = polymerase chain reaction
• **Purpose** = amplify DNA
• Inventor = Kary B. Mullis
• **Year** = 1983
• **Nobel Prize** = 1993
• **Enzyme** = Taq polymerase
• Source = Thermus aquaticus
• Property = temperature-tolerant; stable at near-boiling temperatures
• Components = template DNA; dNTPs; primers; Taq polymerase; buffer
• **Instrument** = thermocycler
• Steps = denaturation; primer annealing; extension

Detailed Explanation

Polymerase Chain Reaction (PCR)

• The technique, which is used to amplify (clone) a single gene or a piece of DNA into thousands to millions of copies by means of in-vitro replication process is called polymerase chain reaction (PCR).
• In this technique, DNA polymerase is compelled to polymerize (polymerase reaction) a given piece of DNA again and again, so that multiple copies are produced, thus, the technique is known as polymerase chain reaction (PCR).

History

• PCR Technique was invented by Kary B. Mullis in 1983.
• Later on he was awarded the Nobel Prize in Chemistry in 1993.

Taq Polymerase

• A special DNA polymerase, the Taq polymerase is used in PCR technique.
• It is a specialized temperature-tolerant enzyme isolated from Thermus aquaticus, a bacterium found in hot springs.
• This enzyme is stable and active at near-boiling temperatures.

Components of PCR

• Template DNA (DNA to be amplified)
• Free nucleotides (deoxyribonucleoside triphosphates or dNTPs)
• Primers
• Taq polymerase

These are diluted in a suitable buffer to make PCR reaction mixture.

Thermocycler

• The PCR mixture is placed in an instrument called thermocycler or PCR machine.
• Thermocycler regulates the temperature during various steps of PCR reaction according to the need.

Mechanism of PCR Reaction

• Denaturation
• Primer annealing
• 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

• 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 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 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.

• 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.

Summary Table of PCR Steps

Summary of PCR Components

Intermediate Level

PCR Steps

PCR Components

Taq Polymerase

Formula / Mathematical Form

Not applicable to this topic.

Solved Example

Not applicable to this topic (descriptive topic).

Important Points

• 1. PCR is a technique to amplify DNA.
• 2. It produces thousands to millions of copies.
• 3. It is an in-vitro replication process.
• 4. PCR was invented by Kary B. Mullis in 1983.
• 5. He was awarded Nobel Prize in 1993.
• 6. Taq polymerase is used in PCR.
• 7. Taq polymerase is temperature-tolerant.
• 8. Taq polymerase is isolated from Thermus aquaticus.
• 9. Taq polymerase is stable at near-boiling temperatures.
• 10. PCR requires template DNA, dNTPs, primers, Taq polymerase, and buffer.
• 11. PCR is performed in a thermocycler.
• 12. Thermocycler regulates temperature.
• 13. PCR has three main steps: denaturation, primer annealing, extension.
• 14. Initial denaturation is at 94-95°C for 4-5 minutes.
• 15. Cyclic denaturation is at 94°C for 1 minute.
• 16. Primer annealing is at 55-65°C for 2 minutes.
• 17. Cyclic extension is at 72°C for 1-2 minutes.
• 18. Final extension is at 72°C for 5-7 minutes.
• 19. PCR products are stored at 4°C.
• 20. PCR is used in research, medicine, and forensic science.

Remember

• - PCR = polymerase chain reaction
• - **Purpose** = amplify DNA
• - Inventor = Kary B. Mullis
• - **Year** = 1983
• - **Nobel Prize** = 1993
• - **Enzyme** = Taq polymerase
• - Source = Thermus aquaticus
• - Property = temperature-tolerant; stable at near-boiling temperatures
• - Components = template DNA; dNTPs; primers; Taq polymerase; buffer
• - **Instrument** = thermocycler
• - Steps = denaturation; primer annealing; extension

Common Mistakes

• 1. Confusing PCR with DNA replication — PCR is in-vitro; DNA replication is in-vivo.
• 2. Mixing up the temperatures — Denaturation: 94°C; annealing: 55-65°C; extension: 72°C.
• 3. Forgetting the role of Taq polymerase — Synthesizes new DNA.
• 4. Mixing up the source of Taq polymerase — Thermus aquaticus.
• 5. Forgetting the inventor — Kary B. Mullis.
• 6. Mixing up the steps — Denaturation, annealing, extension.
• 7. Thinking PCR is a single step — PCR has multiple steps.
• 8. Mixing up the components — Template DNA, dNTPs, primers, Taq polymerase, buffer.
• 9. Forgetting the role of thermocycler — Regulates temperature.
• 10. Believing PCR is not important — PCR is a fundamental technique in molecular biology.

Quick Revision

The technique, which is used to amplify (clone) a single gene or a piece of DNA into thousands to millions of copies by means of in-vitro replication process is called polymerase chain reaction (PCR). In this technique, DNA polymerase is compelled to polymerize (polymerase reaction) a given piece of DNA again and again, so that multiple copies are produced, thus, the technique is known as polymerase chain reaction (PCR). PCR Technique was invented by Kary B. Mullis in 1983; later on he was awarded the Nobel Prize in Chemistry in 1993. A special DNA polymerase, the Taq polymerase is used in PCR technique, which is specialized temperature-tolerant enzyme isolated from Thermus aquaticus, a bacterium found in hot springs. This enzyme is stable and active at near-boiling temperatures. In order to perform PCR, template DNA (DNA to be amplified), free nucleotides (deoxyribonucleoside triphosphates or dNTPs), primers and Taq polymerase are diluted in a suitable buffer to make PCR reaction mixture. The PCR mixture is placed in an instrument called thermocycler or PCR machine. Thermocycler regulates the temperature during various steps of PCR reaction according to the need. 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. 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 = polymerase chain reaction
• Purpose = amplify DNA
• Inventor = Kary B. Mullis
• Year = 1983
• Nobel Prize = 1993
• Enzyme = Taq polymerase
• Source = Thermus aquaticus
• Property = temperature-tolerant; stable at near-boiling temperatures
• Components = template DNA; dNTPs; primers; Taq polymerase; buffer
• Instrument = thermocycler
• Steps = denaturation; primer annealing; extension
• Warning: PCR is an in-vitro process; DNA replication is in-vivo

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• 10. Biotechnology

• Topic:
• 10.6 MECHANISM OF PCR REACTION

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