The Steps of PCR - Online Tutor, Practice Problems & Exam Prep

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 incorporating nucleotides, 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 is going to occur at high temperatures. The second step within each PCR cycle is going to be annealing, which is going to occur at low temperatures. The third and final step within each PCR cycle is going to be extension, which is going to occur at moderate temperatures. As we move forward in our course, we're going to talk about each of these three steps within each PCR cycle in more detail. But these three steps are going to be repeated in each PCR cycle, generating an exponentially growing number of DNA molecules.

Below in this image, what we're showing you are the steps of PCR within just one cycle. At the very beginning, we're of course going to have our reactants and all of the four components that we talked about in our previous lesson videos, which includes the template DNA, the PCR primers, the heat-resistant or thermostable DNA polymerase, Taq polymerase, and also the four nucleotides, the DNA nucleotides. Those would all be part of the reactants within the test tube.

Of course, it's going to include the template DNA, which is the gene of interest, which is what we're focusing on right here. In the very first step of the PCR cycle, we have denaturation, which is going to occur at higher temperatures. The DNA is going to be heated until the DNA denatures. The denaturation of the DNA is going to separate the two strands of DNA. You can see that we have these flames here indicating the increase in temperature that is going to denature the DNA and separate the two strands. Thus, allowing each of those DNA strands to serve as a template.

In the second step of each PCR cycle, what we have is the process of annealing. This is going to occur at cooler temperatures. The DNA primers are going to bind with the single-stranded DNA at these cooler temperatures. Now, the DNA primers have bound to the DNA and they are oppositely oriented and facing towards each other in terms of their 5' and 3' ends pointing towards each other.

Then, we have the process of extension, which is going to occur at moderate temperatures. The heat-resistant or the thermostable DNA polymerase called Taq polymerase is going to build new DNA strands. It's going to be extending off of the primers in that 5' to 3' direction for both strands, and it will be building and amplifying the DNA.

What we end up with are the products at the end of the first cycle of PCR. The gene of interest is going to have been amplified, at the end of the PCR cycle. You can see that now we have two identical copies of the original gene of interest. Again, these steps, these PCR steps occur in cycles. These two here 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 here 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

This video we're going to focus on the first step of PCR, which is denaturation. In the very first step of PCR cycle, heat is going to be used to denature the DNA. We're going to 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 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 is going to help break all of the hydrogen bonds between complementary base pairs in the DNA. Breaking those hydrogen bonds is going to separate 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. The Taq polymerase is able to withstand extremely high temperatures, but it's also able to withstand rapid temperature decreases as well. We're going to 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.

In the very first step of PCR, what we have is denaturation. Within our test tube, we're going to have our DNA, and we'll need to raise the temperature, which you can see these flames here are going to raise the temperature, and the heat is going to denature. So the heat denatures the complementary DNA strands, and denaturing the DNA really just means that the hydrogen bonds are going to be broken and the DNA is going to be forming its single-stranded form. So you can see we have two single-stranded DNA molecules here.

Again, within this test tube, we need to ensure that there is a thermostable polymerase. Over here, we have our thermostable polymerase here. You can see he’s got this name tag that says, hello, my name is Taq, because this is Taq polymerase. Notice that Taq polymerase has no problems with the heat. The 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. So 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 step, the DNA primers actually anneal to the heat-denatured single-stranded DNA. The temperature is lowered to about 55 degrees Celsius. If you recall in step number 1, the temperatures were as high as 95 degrees Celsius, so there is a significant drop from 95 degrees Celsius down to about 55 degrees Celsius, which is important for lowering the temperature. This lowering of the temperature allows for complementary base pairing between the DNA primers and the single-stranded DNA.

Taq polymerase remains inactive at these cooler temperatures because the temperatures are too cool for it to synthesize DNA. When we look at our image on the left hand side, at the second step, annealing, you can see that the temperatures are cooled. 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 cooled so that the DNA primers can anneal to the DNA as we see here. Notice that they are annealed on different DNA strands, and they are annealed facing toward each other in terms of their 5-prime and 3-prime ends. This one goes from left to right, and this one goes from right to left, facing toward 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 the temperature of 55 degrees Celsius is just too cold for the Taq polymerase to work. Notice the Taq polymerase is saying, "brrrr, I can't work like this," and he's all cold, and he can only work when we increase those temperatures just a little to the right temperatures. This concludes our brief introduction to the second step of PCR, the annealing of the DNA primers. We'll be able to talk about the 3rd 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 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 prime to 3 prime 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.

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.

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.

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.

Here’s what students ask on this topic:

What are the steps of PCR and their respective temperatures?

The Polymerase Chain Reaction (PCR) consists of three main steps: denaturation, annealing, and extension. During denaturation, the DNA is heated to approximately 95°C to break the hydrogen bonds between the DNA strands, 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 increased to around 72°C, which is the optimal temperature for Taq polymerase to synthesize new DNA strands by incorporating nucleotides. This cyclical process is repeated multiple times to exponentially amplify 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 the denaturation step (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 success of PCR.

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 breaks the hydrogen bonds between the complementary base pairs in the double-stranded DNA, resulting in the separation of the DNA into single strands. These single-stranded DNA molecules serve as templates for the subsequent steps of PCR. The denaturation step is essential for making the DNA accessible for primer binding and subsequent DNA synthesis.

How does the annealing step in PCR work?

In the annealing step of PCR, the temperature of the reaction mixture is lowered to about 55°C. This cooler temperature allows the DNA primers to bind, or anneal, to their complementary sequences on the single-stranded DNA templates. The primers are short sequences of nucleotides that provide a starting point for DNA synthesis. Proper annealing is crucial for the specificity of PCR, as it ensures that the primers bind only to the target DNA sequences.

What is the role of the extension step in PCR?

The extension step in PCR occurs at around 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' end of each primer. The enzyme extends the primers in the 5' to 3' direction, creating new double-stranded DNA molecules. This step results in the amplification of the target DNA, doubling the amount of DNA with each cycle. The extension step is critical for generating sufficient quantities of the target DNA for analysis.