The Steps of PCR: Videos & Practice Problems

The polymerase chain reaction (PCR) consists of three key steps: denaturation, annealing, and extension. During denaturation, DNA is heated to approximately 95°C, breaking hydrogen bonds and forming single strands. In the annealing step, the temperature is lowered to about 55°C, allowing DNA primers to bind to the single-stranded DNA. Finally, at 72°C, Taq polymerase synthesizes new DNA strands by extending from the primers, resulting in two identical copies of the original DNA. This cyclical process amplifies the target DNA exponentially, making PCR a vital tool in molecular biology.

The Steps of PCR

In this video, we're going to talk about the steps of PCR or polymerase chain reaction. PCR is actually a cyclical process that occurs in cycles, where each of the cycles is going to have 3 steps that we have numbered down below. The very first step within each cycle is the step of denaturation, which occurs at high temperatures. The second step within each PCR cycle is annealing, occurring at low temperatures. The third and final step within each PCR cycle is extension, occurring at moderate temperatures. As we move forward in our course, we're going to talk about each of these 3 steps within each PCR cycle in more detail. These three steps are repeated in each PCR cycle, generating an exponentially growing number of DNA molecules. Below in this image, we are showing you the steps of PCR within just one cycle. At the very beginning, we have our reactants and all of the 4 components that we talked about in our previous lesson videos, which include the template DNA, the PCR primers, the heat-resistant or thermostable DNA polymerase, Taq polymerase, and also the 4 nucleotides, the DNA nucleotides. These are all part of the reactants within the test tube. It includes the template DNA, which is the gene of interest we are focusing on right here.

In the very first step of the PCR cycle, we have denaturation, which occurs at higher temperatures. The DNA is heated until the DNA denatures, separating the two strands of DNA. You can see we have these flames here indicating the increase in temperature that will denature the DNA and separate the two strands, breaking the hydrogen bonds. In the second step of each PCR cycle, we have the process of annealing. This occurs at cooler temperatures. The DNA primers bind with the single-stranded DNA at these cooler temperatures. You can see that the DNA primers have bound to the DNA and are oppositely oriented, with their 5' and 3' ends pointing towards each other.

Then, we have the process of extension, which occurs at moderate temperatures. The heat-resistant or the thermostable DNA polymerase, called Taq polymerase, builds new DNA strands. It extends off of the primers in the 5' to 3' direction for both strands, building and amplifying the DNA. At the end of the first cycle of PCR, the gene of interest has been amplified. You can see that we now have 2 identical copies of the original gene of interest. These steps of PCR occur in cycles repeatedly. These new copies would be subject to the same process: denaturation, annealing, and extension over and over again in these sets of cycles.

We'll be able to talk about each of these steps within each cycle in more detail as we move forward in our course. But for now, this concludes our brief introduction to the steps of PCR: denaturation, annealing, and extension, and I'll see you all in our next video.

1) Denaturation

In this video, we're going to focus on the first step of PCR, which is denaturation. In the very first step of the PCR cycle, heat is used to denature the DNA. We heat denature the double-stranded DNA in order to convert the DNA into its single-stranded form. Each of the single-stranded DNA molecules can serve as a template for a new molecule of DNA. The temperature of the PCR mixture is going to be increased to about 95 degrees Celsius, which is very near boiling temperatures. Increasing the temperature to 95 degrees Celsius helps break all of the hydrogen bonds or the H bonds between complementary base pairs in the DNA. Breaking these hydrogen bonds separates the double-stranded DNA into its single-stranded form.

It's very important that a special thermostable DNA polymerase, such as Taq polymerase, is used in the PCR mixture. This Taq polymerase does not denature at the high temperatures used in PCR. Taq polymerase is able to withstand extremely high temperatures, but it is also able to withstand rapid temperature decreases as well. We will see rapid temperature decreases in the second step of PCR. However, it's also important to note that although Taq polymerase can withstand high temperatures and rapid temperature decreases, it does not actually synthesize DNA unless it is at an ideal temperature. This will be important once we get to the third step of PCR.

In the very first step of PCR, we have denaturation. Within our test tube, we need to raise the temperature, and the heat denatures the complementary DNA strands. Denaturing the DNA means that the hydrogen bonds are broken and the DNA forms its single-stranded state. You can see we have two single-stranded DNA molecules here. Again, within this test tube, it is necessary to ensure that there is a thermostable polymerase. Over here, what we have is our thermostable polymerase with a name tag that says, "Hello, my name is Taq," because this is Taq polymerase. Notice that heat never bothered him anyway. Taq polymerase is going to be very important to have and use during PCR.

This here concludes our introduction to the first step of PCR, denaturation. As we move forward, we'll be able to talk about the second and third steps of PCR. I'll see you all in our next video.

2) Annealing

In this video, we're going to talk more about the second step of each PCR cycle, which is annealing. In this second step, the DNA primers are actually going to anneal to the heat-denatured single-stranded DNA. The temperature is going to be lowered to about 55 degrees Celsius. If you recall in step number 1, the temperatures were as high as 95 degrees Celsius. This lowering of the temperature allows for complementary base pairing to occur between the DNA primers and the single-stranded DNA itself.

Taq polymerase remains inactive at these cooler temperatures because the temperatures are just too cool for it to synthesize DNA. When we take a look at our image on the left-hand side, at the second step, annealing, you can see that the cooler temperatures allow the DNA primers to anneal to the DNA. After the first step of PCR, which is denaturation, we know that we've generated single-stranded DNA, this heat-denatured DNA. In the second step, the temperatures are cold so that the DNA primers can anneal to the DNA, as we see here. Notice that they are annealed on different DNA strands and are annealed facing towards each other in terms of their 5' and 3' ends. This one goes from left to right, and this one goes from right to left, facing towards each other.

Now that these DNA primers have been annealed, in the third step, they can be extended. However, it cannot be extended here in the second step because these temperatures of 55 degrees Celsius are just too cold for the Taq polymerase to work. Notice the Taq polymerase is saying, "brrr, I can't work like this." He's all cold, and he can only work when we increase those temperatures just a little bit to the right temperatures. This here concludes our brief introduction to the second step of PCR, annealing of the DNA primers. We'll be able to talk about the third and final step of PCR in our next video.

3) Extension

In this video, we're going to talk about the 3rd and final step of each PCR cycle, which is extension. And so the final step of PCR, which is this third step, is the extension of the new DNA strand by the thermostable Taq polymerase. And so the temperature in the third step is going to be changed and increased from 55 degrees Celsius in the second step to about 72 degrees Celsius here in the third step. And this 72 degrees Celsius is an ideal temperature for the activity of the Taq polymerase. And so the Taq polymerase is going to incorporate deoxyribonucleotides and or those DNA nucleotides and use them to extend the DNA primers on each strand of the DNA, making 2 identical copies of the DNA. And so if we take a look at our image down below at this third step here of the PCR cycle extension, what you'll notice is that over here on the left, we're starting off with what we ended with in the second step, which is the annealing of those DNA primers. And so here in the second step, the temperature is going to be increased to the ideal temperature for Taq polymerase. And so the Taq Polymerase is able to incorporate the DNA nucleotides, the free DNA nucleotides, and use those free DNA nucleotides to extend each of the primers. And so you can see that the extension of the primers is always going to be in the 5' to 3' direction. And so the Taq polymerase will extend this in this direction, and the Taq polymerase here will extend this primer in this direction from right to left. And so the Taq polymerase extends the new strands of DNA until we have the template DNA which has been replicated. So the template DNA has been replicated here. And so you can see that originally we now have 2 identical copies of the original template DNA. And so, that is going to conclude this 3rd and final step of each PCR cycle. And so after the third step has been completed, then it can go back into a new cycle of PCR, where each of these products from the 1st cycle can be used as reactants in the second cycle and so on. And so this here concludes our introduction to the 3rd step of PCR extension. And we'll be able to get some practice applying all of the concepts that we've talked about as we move forward in our course. So I'll see you all in our next video.

Which of the following correctly lists the steps in order for one cycle of the polymerase chain reaction (PCR)?

• A. Denature DNA; Add DNA polymerase; Anneal primers; Add dNTPs; Extend primers.
• B. Anneal primers; Denature DNA; Extend primers.
• C. Extend primers; Anneal primers; Denature DNA.
• D. Denature DNA; Anneal primers; Extend primers utilizing a thermostable DNA polymerase.

Answer: (D)

Why is a DNA polymerase from a thermophilic bacterium used in PCR?

• A. The enzyme makes DNA that is extremely similar to human DNA.
• B. It is cheaper to obtain from live microorganisms than producing the enzyme in a lab.
• C. This thermophile's DNA polymerase does not require primers to begin DNA synthesis.
• D. This thermophile's DNA polymerase can withstand high temperatures that denature most proteins.

Answer: (D)

PCR is known for its power of amplifying a target DNA sequence at a high speed. Each cycle can double the 
number of DNA molecules (target sequence). Which of the following is CORRECT regarding PCR?

• A. In order to make 10 copies of the DNA, you need at least 5 cycles of PCR.
• B. Helicase is required in order to separate the two strands in PCR.
• C. Dideoxynucleotides are used in PCR.
• D. DNA primers are needed in PCR.
• E. All of the above.

Answer: (D)

If you start with one double-stranded DNA molecule and you perform SIX cycles of PCR, how many double
stranded copies of the DNA will you have?

• A. 6.
• B. 8.
• C. 16.
• D. 32.
• E. 64.

Answer: (E)

Here’s what students ask on this topic:

What are the three main steps of PCR?

The three main steps of PCR (Polymerase Chain Reaction) are denaturation, annealing, and extension. During denaturation, the DNA is heated to approximately 95°C, causing the hydrogen bonds between the DNA strands to break, resulting in single-stranded DNA. In the annealing step, the temperature is lowered to about 55°C, allowing DNA primers to bind to the single-stranded DNA. Finally, during the extension step, the temperature is raised to around 72°C, which is the optimal temperature for Taq polymerase to synthesize new DNA strands by incorporating nucleotides. These steps are repeated in cycles, leading to exponential amplification of the target DNA.

Why is Taq polymerase used in PCR?

Taq polymerase is used in PCR because it is a thermostable enzyme that can withstand the high temperatures required for DNA denaturation (around 95°C). Unlike other DNA polymerases, Taq polymerase does not denature at these high temperatures, making it ideal for the PCR process. Additionally, Taq polymerase is efficient at synthesizing DNA at the optimal temperature of 72°C during the extension step, ensuring accurate and rapid DNA replication. Its ability to function under these conditions is crucial for the cyclical nature of PCR, which involves repeated heating and cooling.

What happens during the denaturation step of PCR?

During the denaturation step of PCR, the reaction mixture is heated to approximately 95°C. This high temperature causes the hydrogen bonds between the complementary base pairs of the double-stranded DNA to break, resulting in the separation of the DNA into single strands. This step is essential because it provides single-stranded DNA templates for the subsequent annealing and extension steps. The denaturation step ensures that each cycle of PCR starts with single-stranded DNA, allowing for the exponential amplification of the target DNA sequence.

What is the purpose of the annealing step in PCR?

The purpose of the annealing step in PCR is to allow the DNA primers to bind to the single-stranded DNA templates. During this step, the temperature is lowered to about 55°C, which is optimal for the primers to form hydrogen bonds with their complementary sequences on the single-stranded DNA. This binding is crucial because the primers provide the starting point for DNA synthesis in the next step, extension. Proper annealing ensures that the primers are correctly positioned for Taq polymerase to extend the DNA strands, leading to accurate amplification of the target sequence.

How does the extension step in PCR work?

In the extension step of PCR, the temperature is raised to approximately 72°C, which is the optimal temperature for Taq polymerase activity. During this step, Taq polymerase synthesizes new DNA strands by adding nucleotides to the 3' ends of the annealed primers. The enzyme extends the primers in the 5' to 3' direction, creating new complementary DNA strands. This process results in the formation of two identical copies of the original DNA template. The extension step is repeated in each cycle of PCR, leading to the exponential amplification of the target DNA sequence.