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Polymerase Chain Reaction (PCR): Amplifying DNA

Polymerase Chain Reaction (PCR) stands as one of the most transformative techniques in modern biology, revolutionizing the way scientists manipulate and study DNA. Developed in the 1980s by Nobel laureate Kary Mullis, PCR provides researchers with a powerful tool to amplify specific segments of DNA, making millions of copies in a short period. Its impact spans across various fields, from molecular biology and genetics to medicine, forensics, and biotechnology. By unlocking the potential to replicate and analyze DNA with unprecedented precision and efficiency, PCR has propelled countless discoveries, advancements, and innovations, shaping our understanding of genetics and influencing applications ranging from disease diagnosis to evolutionary biology.

 

TL; DR

  • Polymerase chain reaction is a technique that can create millions of copies of DNA from a single DNA molecule.
  •  PCR requires a special, heat-stable enzyme (Taq polymerase).
  • PCR has broad applications in science/medicine research, diagnostics and therapeutics.

Important Discoveries Before PCR

The story begins with the quest to decipher the genetic code, the blueprint of life encoded within our DNA. In the 1950s and 1960s, scientists made groundbreaking discoveries that laid the foundation for PCR. One such discovery was the elucidation of the structure of DNA by James Watson and Francis Crick in 1953, revealing its double-helix structure and the complementary base-pairing rules.

Fast forward to the 1970s, when scientists discovered enzymes called DNA polymerases, which play a crucial role in DNA replication by synthesizing new DNA strands using existing templates. Another key breakthrough came with the discovery of a heat-stable DNA polymerase in the bacterium Thermus aquaticus (Taq), which thrives in hot springs.

What is Polymerase Chain Reaction?

Armed with these foundational discoveries, Nobel laureate Kary Mullis invented PCR in 1983, forever changing the landscape of molecular biology. PCR is a technique that allows scientists to amplify small segments of DNA exponentially, making millions of copies in a short amount of time.

So, how does PCR work? It’s like a molecular photocopier that selectively copies a specific region of DNA over and over again. Here’s a simplified breakdown of the process:

      1. Denaturation: The double-stranded DNA template is heated to around 95°C, causing the two strands to separate and unwind, forming single-stranded DNA molecules.
      2. Annealing: The temperature is lowered to around 50-65°C, allowing short DNA sequences called primers to bind to complementary sequences on the single-stranded DNA template.
      3. Extension: The temperature is raised to around 72°C, and a heat-stable DNA polymerase, such as Taq polymerase, synthesizes new DNA strands by extending the primers along the template DNA.

 

By repeating these cycles of denaturation, annealing, and extension multiple times (typically 20-40 cycles), the targeted DNA sequence is amplified exponentially, resulting in millions of copies that can be easily detected and analyzed.

Definition – Polymerase

In biology, enzyme are easily spotted by the suffix –ase. The word they’re affixed to denotes the substrate on which they act. Polymerase is therefore an eznyme that acts on a polymer; the polymer being DNA. 

Author: Enzoklop | License: CC BY-SA 3.0

Denaturation (Step 1)

Purpose: separate double standed DNA molecule into two single stranded DNA molecules.

  1. Temperature = ~96ºC.
  2. As a result, the hydrogen bonds in the DNA double strand break and the molecule separates into two separate DNA single strands. (Excuse the alliteration!)

Annealing (Step 2)

 Purpose: allow for primers to attach to each single stranded DNA molecule.
 
  1. Temperature = ~68ºC.
  2. This will allow primers to attach to complimentary sequences on each of the single stranded DNA.
 

Elongation (Step 3)

Purpose: extend primers by adding nucleotides thereby creating a copy of the original molecule.
 
  1. Temperature = 72ºC.
  2. Taq polymerase will interact with the DNA-primer complex and begin to add nucleotides to the 3′ end of the primer. These nucleotides will be complimentary to whatever nucleotides are on the DNA strand.

What Do You Need for a PCR Experiment?

Ingredients

      • ✔ DNA sample – this is the DNA we want to copy.
      • ✔ Taq polymerase – enzyme that polymerizes DNA copies from the DNA template.
      • ✔ Primers – short nucleotide sequence that attaches to DNA template.
      • ✔ Free-floating nucleotides (adenine, guanine, cytosine, thymine).
      • ✔ PCR tube – to contain our PCR mixture.
      •  

The Experiment

    • ✔ PCR mixture
    • ✔ Thermocycler – this machine will cycle the temperature of our reaction.
    •  

After all ingredients are added to our PCR tube, we can place the mixture into a machine called a thermocycler – or thermal cycler. As it’s name suggests, the thermocycler will adjust the temperature between three or four temperature settings that are required for each step in the PCR reaction. One cycle of PCR has just three steps and takes just a couple of minutes to complete. At the end of a cycle, the concentration of our DNA would have doubled.

Importance of PCR in Science & Society

The invention of PCR has had profound implications across various fields, revolutionizing scientific research, medical diagnostics, forensic analysis, and beyond. Here are just a few ways PCR has impacted our world:

Medical Diagnostics: PCR is used to detect and diagnose infectious diseases, genetic disorders, and cancer by amplifying specific DNA sequences associated with these conditions. It allows for early detection, personalized treatment strategies, and monitoring of disease progression.

Forensic Analysis: PCR is employed in forensic science to analyze DNA evidence collected from crime scenes, identify suspects, and exonerate the innocent. It has played a crucial role in solving countless criminal cases and ensuring justice is served.

Genetic Engineering: PCR is a cornerstone technique in genetic engineering and biotechnology, enabling the manipulation and modification of DNA for various purposes, such as gene cloning, sequencing, and gene editing using technologies like CRISPR.

Paleontology and Archaeology: PCR has revolutionized the fields of paleontology and archaeology by allowing scientists to extract and amplify ancient DNA from fossils and archaeological artifacts. This has provided valuable insights into the evolutionary history of species and human populations.

Polyermase Chain Reaction FAQs & Review

A single cycle of PCR – denaturation, annealing and elongation – should double your concentration of DNA molecules.

Somewhat controversial figure, Kary Mullis invented PCR in 1983. Mullis claims to have come up with the idea during a car ride near his country home in Northern California. PCR is such an influential process, Mullis would earn the 1993 Nobel Prize in Chemistry for it’s invention.

This particular variant of polymerase is produced by the species Thermus aquaticus. These bacteria thrive in hot temperatures (~65 C). Therefore, the proteins produced by these bacteria are stable within this temperature range. Since the denaturation phase of PCR requires temperatures close to 100 C, Taq polymerase is a great candidate for the job. Other polymerases are likely to denature at these temperatures.

The goal of polymerase chain reaction is to turn a single DNA molecule into millions of copies. Each step in a cycle of PCR does something in particular to allow for the replication process.

The denaturation step separates the double stranded DNA into a two single stands of DNA. Without this step, there would not be a way to read and replicate each strand.

The annealing step allows for primers, to attach to each template. Without this step, there would not be a way to allow for Taq polymerase to attach to the short double stranded sequence of DNA.

The elongation step allows Taq polymerase to read the template strand and add complementary nucleotides to the elongating strand. Without this step, we would not create our double stranded copy.

Key Terms

    • Polymerase Chain Reaction
    • Taq Polymerase
    • Primer
    • Nucleotides
    • Denaturation
    • Annealing
    • Elongation
    • Thermocycler

 

  1. Zedalis, Julianne, and John Eggebrecht. “DNA Structure and Sequencing.” Biology for AP® Courses, OpenStax, https://openstax.org/books/biology-ap-courses/pages/17-1-biotechnology. License: CC BY 4.0 License Terms: Edited & Adapted | Access for free at https://openstax.org/books/biology-ap-courses/pages/1-introduction
  2. Enzoklop. “File:Polymerase Chain Reaction.svg.” Wikimedia Commons, 23 Sept. 2019, https://commons.wikimedia.org/w/index.php?title=File:Polymerase_chain_reaction.svg&oldid=221098147. License: CC BY-SA 3.0 License Terms: No Edits were made.
  3. CanalDivulgación. “PCR – Polymerase Chain Reaction (IQOG-CSIC).” YouTube, 4 Winter 2014, https://www.youtube.com/watch?v=iQsu3Kz9NYo&t=34s.