Polymerase Chain Reaction (PCR): Copying DNA
Polymerase chain reaction (PCR) is a technique that scientists use to amplify DNA for further analysis. Researchers use PCR for many purposes in laboratories, such as cloning gene fragments to analyze genetic diseases, identifying contaminant foreign DNA in a sample, and amplifying DNA for sequencing. More practical applications include determining paternity and detecting genetic diseases.
Why PCR is so Important?
Most of the time when we want to work with DNA, we don’t have a lot of it available and most experimental procedures require more DNA. The problem of ‘not enough DNA’ remained a major issue in science, until Kary Mullis invented polymerase chain reaction (PCR). PCR is a laboratory procedure that can take small quantities of DNA – even just a single molecule – and within a couple hours, copy that molecule millions of times! The technique has completely changed modern science and medicine. It allowed scientists and physicians the ability to manipulate DNA in a fast, reliable way that was not previously possible. The technique was so influential that Mullis would earn several prestigious awards including the 1993 Nobel Prize in Chemistry, which he shared with Michael Smith.
What Do You Need for a PCR Experiment?
- 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.
- 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.
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.
One Cycle of PCR: Step-by-Step
Denaturation (Step 1)
Purpose: separate double standed DNA molecule into two single stranded DNA molecules.
- Temperature = ~96ºC.
- 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)
- Temperature = ~68ºC.
- This will allow primers to attach to complimentary sequences on each of the single stranded DNA.
Elongation (Step 3)
- Temperature = 72ºC.
- 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.
Taq Polymerase - A Very Special Polymerase
Taq polymerase is a special type of polymerase named from the bacteria that produces it Thermus aquaticus. This bacteria is thermophilic and grows best at temperatures around 150°F, which is why it was first discovered near the hot springs of Yellowstone National Park. If you look at the image, that bright orange ring around the hot spring is Thermus aquaticus.
At these temperatures, most proteins denature. However, since this organism thrives in this environment, it produces proteins that are heat-stable. The fact that the polymerase produced by Thermus aquaticus is heat-stable is what allows for it to be successfully in a PCR reaction; polymerases from other organisms would denature during the elevated temperatures required for the denaturation of DNA.
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.
- Polymerase Chain Reaction
- Taq Polymerase
- 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
- 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.
- CanalDivulgación. “PCR – Polymerase Chain Reaction (IQOG-CSIC).” YouTube, 4 Winter 2014, https://www.youtube.com/watch?v=iQsu3Kz9NYo&t=34s.