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Polymerase Chain Reaction: Definition & Steps

Instructor: Rachel Torrens
Have you ever wondered if you could make millions of copies of something in a matter of minutes. We're not discussing a copy machine, but rather a fascinating laboratory procedure called polymerase chain reaction. Learn all about it in this lesson!

What Is the Polymerase Chain Reaction?

Humans have long tried to improve upon nature, and one attempt to do so is with polymerase chain reaction, commonly abbreviated PCR. This procedure is a man-made version of DNA replication carried out in a lab, and it rapidly generates many copies of DNA. Before we delve into the details of PCR and how scientists use it to mimic natural DNA replication, let's brush up on the way Mother Nature duplicates DNA.

Natural DNA Replication

Remember DNA, or deoxyribonucleic acid, is composed of four building blocks, or nucleotides: guanine, cytosine, adenine, and thymine. Guanine bonds to cytosine while adenine bonds to thymine, forming a double helix. Another way to picture DNA's structure is to envision a ladder--now grab a hold of both ends and twist!

The double helix of DNA. Notice the complementary nucleotides--guanine with cytosine, and adenine with thymine.
DNA double helix with complementary nucleotides

The first step necessary to copy a strand of DNA is to untwist and separate the two strands. This is accomplished by the enzyme helicase. Next, once the strands are separated, RNA primase (another enzyme) attaches a primer. The primer tells the cell's building crew (yet another enzyme known as DNA polymerase) where exactly to start replicating the DNA. Lastly, the polymerase begins adding complementary nucleotides to the single-stranded DNA, thereby creating a new double helix DNA molecule!

Steps of Polymerase Chain Reaction

DNA replication is a complicated procedure. However, scientists have successfully found a way to carry it out in the controlled environment of a test tube. What goes in that test tube is very important. The scientist adds the DNA or template DNA, followed by a PCR buffer. The PCR buffer is a liquid that maintains optimal pH and salt concentrations, even when other ingredients are added.

With these ingredients ready, we can begin the copying process. Recall that there are three steps in natural DNA replication. During PCR, these are called denaturing, annealing, and extension. Let's look at each step in detail:

  1. Denaturation: In order to make a copy of the DNA, the strands must be separated. Instead of using helicase like Mother Nature does, scientists use heat to break the bonds between complementary nucleotides. The DNA is heated to approximately 95 degrees Celsius for 30 seconds. This breaks the bonds and causes the DNA to unwind.
  2. Annealing: The next step is to add a primer that will serve to flag down the building crew--the polymerase--so it knows where to start copying. To accomplish this, scientists add PCR primer, a small piece of DNA that dictates which part of the double helix will be copied. During this step, the test tube is lowered to 50 degrees Celsius for 30 seconds.
  3. Extension: Now that we've unwound and primed, all that's left is to do is add on new complementary nucleotides. In nature, DNA polymerase fulfills this duty. But for PCR, scientists use Taq polymerase, a thermostable polymerase (it comes from an organism found in hot springs, which means it can tolerate the hot temperatures required). Taq polymerase adds the complementary nucleotides to the single-stranded DNA, forming a new, duplicate double helix. This step takes about 60 seconds and requires the test tube temperature to be raised to 72 degrees Celsius.

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