10.5 Polymerase Chain Reaction (PCR)

The Polymerase Chain Reaction (PCR) is a revolutionary molecular biology technique used for the in-vitro amplification (cloning or making many copies) of a specific segment of DNA. It can generate thousands to millions of copies of a particular DNA sequence from a very small initial amount. The technique was invented by Kary B. Mullis in 1983.

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Core Principle

PCR mimics the natural process of DNA replication in a test tube. It uses a series of temperature changes to control the separation and synthesis of DNA strands, leading to an exponential increase in the number of copies of the target DNA sequence.

Essential Components for a PCR Reaction

The reaction is set up in a tube with a specific mixture of components:

• Template DNA: The original DNA molecule containing the sequence to be amplified.
• Primers: Short, single-stranded DNA sequences (one forward, one reverse) that are complementary to the start and end of the target sequence. They provide a starting point for DNA synthesis.
• Taq Polymerase: A special, heat-tolerant DNA polymerase enzyme. It is isolated from the bacterium Thermus aquaticus, which lives in hot springs. Unlike most polymerases, Taq polymerase remains stable and active at the high temperatures required for PCR.
• Deoxyribonucleoside triphosphates (dNTPs): The building blocks (A, T, C, G) that Taq polymerase uses to synthesize new DNA strands.
• Buffer: A solution that maintains the optimal pH and provides necessary ions (like $\mathrm{M}{\mathrm{g}}^{2+}$) for the polymerase to function correctly.

The PCR Machine (Thermocycler)

The entire process is automated in an instrument called a thermocycler. This machine can rapidly heat and cool the reaction tubes to the precise temperatures required for each step of the cycle.

The Mechanism of PCR

A complete PCR process involves an initial setup phase, multiple amplification cycles, and a final completion phase.

Non-Cyclic Steps

These steps are performed only once per reaction.

• Initial Denaturation:

  • Temperature: 94-95${}^{\circ }$C
  • Time: 4-5 minutes
  • Purpose: To ensure that all template DNA strands, including complex or GC-rich regions, are fully separated into single strands before the first cycle begins.

• Final Extension:

  • Temperature: 72${}^{\circ }$C
  • Time: 5-7 minutes
  • Purpose: To complete the synthesis of any remaining incomplete DNA strands after the final cycle, ensuring all products are full-length, double-stranded DNA.

• Storage:

  • Temperature: 4${}^{\circ }$C
  • Purpose: To hold the reaction products at a low temperature to preserve the amplified DNA until it can be retrieved.

The PCR Cycle (Repeated 25-35 times)

Each cycle consists of three core steps, and with each cycle, the amount of target DNA doubles.

Applications of PCR

PCR is a fundamental tool in molecular biology with widespread applications:

• Medical Diagnostics: Detecting the DNA of infectious agents like viruses (e.g., HIV, SARS-CoV-2) or bacteria. It is often used in Principles Of Diagnostic Tests→.
• Genetic Testing: Identifying genetic mutations responsible for inherited diseases (e.g., cystic fibrosis, Huntington's disease) and certain cancers.
• Forensics: Amplifying minute amounts of DNA from a crime scene for DNA fingerprinting to identify suspects.
• Research: Cloning genes, studying gene expression, and preparing DNA for DNA sequencing.
• Paternity Testing: Comparing DNA sequences between a child and a potential father.

Possible Questions and Answers

Q: Why can't human DNA polymerase be used in PCR?

A: Human DNA polymerase is not thermostable. It would be destroyed (denatured) during the first denaturation step at 94${}^{\circ }$C and would be unable to synthesize new DNA in subsequent cycles.

Q: Why is heat used to denature DNA in PCR instead of enzymes like DNA helicase?

A: Most enzymes, including DNA helicase, are proteins that would denature at the high temperatures used in PCR. Heat is a simple, efficient, and easily controlled physical method to separate the DNA strands in a laboratory setting. Taq polymerase is the exception because it is adapted to high temperatures.

Q: Why are pre-synthesized primers used in PCR instead of the enzyme primase?

A: Primase synthesizes short RNA primers, not DNA primers, and it does not target specific sequences. PCR requires specific DNA primers to ensure that only the target region of the template DNA is amplified. Using custom-made DNA primers gives the researcher precise control over which gene or DNA segment is copied.