PCR: Reagents Used in Polymerase Chain Reaction

Polymerase chain reaction (PCR) is a biotechnology technique that is used to amplify pieces of DNA. In this lesson, you will learn about five ingredients necessary to perform PCR: template DNA, nucleotides, primers, buffer and Taq polymerase.

PCR in Forensics

Reporter: Professor Pear, your testimony, which helped prove that Colonel Custard was not the Spiral Staircase Killer was fascinating, but it seemed like the defense attorney glossed over the science. Could you please elaborate on how your results helped exonerate the defendant?

Professor Pear: Most definitely, I’m always happy to talk science. My lab was able to demonstrate that Mr. Teal’s DNA was at the scene of the crime. The defense attorney used that evidence to clear her client, Colonel Custard, of the crime and implicate Mr. Teal in the process. Now, the district attorney may charge Mr. Teal with killing Mr. Bones with a lead pipe in the spiral staircase. Even though there were no eyewitnesses or fingerprints to tie Mr. Teal to the scene of the crime, the DNA evidence was inescapable.

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Reporter: And, exactly why is DNA evidence so powerful?

Professor Pear: DNA evidence is so powerful because even minuscule amounts of DNA can be analyzed; and, pretty much every person in the world has a unique DNA signature. PCR is the primary laboratory procedure responsible for making analysis of even small amounts of DNA possible, while DNA sequencing is one way of distinguishing between different people. Even though there were only trace amounts of Mr. Teal’s DNA at the scene of the crime, PCR allowed the crime scene investigators to make enough DNA to run tests to catch him.

Reporter: Wow, that’s amazing! But, what is PCR and how does it work?

Polymerase Chain Reaction

Professor Pear: PCR stands for polymerase chain reaction. It’s a laboratory procedure that can be used to create copies of DNA. It’s basically an artificial version of DNA replication. Replication is the process in which a cell makes copies of DNA.

First, an enzyme called helicase unwinds the DNA double-helix. Then, an enzyme called RNA primase adds a primer to the single-stranded DNA. Finally, an enzyme called DNA polymerase adds nucleotides that are complementary to the single-stranded DNA to create a new double-helix DNA molecule. With PCR, scientists essentially perform DNA replication in a test tube.

Reporter: That seems very complicated.

Professor Pear: Really, it’s not. Let me explain.

Template DNA and Primer

Professor Pear: Scientists basically have figured out a way to mimic each reagent and step that nature devised for DNA replication in a cell. This allows them to amplify segments of DNA for analysis. There are five basic reagents, or ingredients, used in PCR: template DNA, PCR primers, nucleotides, PCR buffer and Taq polymerase.

Remember how I told you that PCR can make more copies of crime scene DNA? That starting DNA is known as the template DNA. Template DNA is the DNA that is amplified during a PCR reaction. Amazingly, scientists can use anything, from saliva to blood, as a source of template DNA in forensic studies. And, because of PCR, only a small amount of DNA is required for analysis.

Just like cellular DNA replication, PCR requires a primer to get started. A PCR primer is a short piece of DNA that identifies the region of DNA that will be amplified during PCR. Just like the primer in a cell, a PCR primer can bind to a specific complementary sequence of DNA. Primers are typically used in pairs, and the DNA between the two primers is amplified during the PCR reaction.

Nucleotides

Professor Pear: DNA molecules consist of monomer subunits called nucleotides. That is, a nucleotide is a subunit of a DNA chain. So, nucleotides are obviously a key part of any PCR reaction.

DNA nucleotides come in four flavors: Guanine, Adenine, Thymine and Cytosine. G forms hydrogen bonds with its complementary base C. And, A forms hydrogen bonds with its complementary base T. Taq polymerase uses these free-floating nucleotides to assemble new copies of the DNA segment being amplified.

The four DNA nucleotides
Four Nucleotides

Reporter: Wait, what the heck is Taq polymerase? You just got through telling me that cells use DNA polymerase.

Taq Polymerase and PCR Buffer

Professor Pear: Oh, I’m sorry. Taq polymerase is a DNA polymerase. It’s just DNA polymerase from a bacterium called Thermus aquaticus. You see, DNA polymerase from Thermus aquaticus is simply abbreviated to Taq polymerase. Taq polymerase serves the same function as human DNA polymerase. It can make copies of DNA prior to cell division.

Like any enzyme, DNA polymerase will function best under certain conditions. PCR buffer is a solution that optimizes conditions like salt concentration and pH to enable Taq polymerase to work efficiently.

Reporter: Wait. If forensic scientists are amplifying human DNA from crime scenes, shouldn’t they be using human polymerase?

Professor Pear: Oh, that’s a good question! One of the amazing things about the DNA code is that it’s universal. Pretty much every organism on the planet uses the same four nucleotides: G, A, T and C. That means that the DNA polymerase from another organism, like Thermus aquaticus, can be used to amplify human DNA. During the PCR process, we need to heat the sample almost to the boiling point.

Reporter: So? Why should that matter?

Professor Pear: Well, human proteins aren’t designed to work at temperatures that high. At a temperature that high, our proteins denature, or unravel. If we tried to use human polymerase in PCR, it would be killed during the boiling step.

Instead, we can use DNA polymerase from Thermus aquaticus because it’s a thermophilic bacterium. Thermo- means ‘heat,’ and -phil means ‘to love.’ So, literally thermophilic means ‘heat-loving.’ Thermus aquaticus lives in hot springs, so its proteins are designed to function at boiling temperatures. Okay, so let me tell you how all these reagents fit together.

Reporter: Wait, wait. Before you go on, let me see if I understand everything you’ve said up to now.

Lesson Summary

Reporter: PCR stands for polymerase chain reaction, which is a laboratory procedure that can be used to create copies of DNA. Template DNA is the DNA that is amplified during a PCR reaction. A PCR primer is a short piece of DNA that identifies the region of DNA that will be amplified during PCR. A nucleotide is a monomer subunit of DNA. Taq polymerase is a heat-stable DNA polymerase that is used in PCR reactions. PCR buffer is a solution that optimizes conditions, like salt concentration and pH, to enable Taq polymerase to work efficiently.

Learning Outcome

After watching this video, you will be able to list and describe the function of the five reagents used in polymerase chain reaction (PCR).

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