Introduction to Polymerase Chain Reaction - Online Tutor, Practice Problems & Exam Prep

Polymerase chain reaction (PCR) is a rapid technique used to amplify specific DNA sequences in a test tube, differing from DNA cloning, which occurs in living cells. PCR is efficient, allowing scientists to generate many identical copies of DNA quickly, often within 1.5 to 2 hours. Key components of PCR include template DNA, two complementary primers, thermostable DNA polymerase (commonly Taq polymerase), and all four deoxyribonucleotides. The amplification process follows cycles, with the number of copies calculated using the formula $2^n$, where n represents the cycle number.

Introduction to Polymerase Chain Reaction

In this video, we're going to begin our introduction to a technique that's called polymerase chain reaction. When a researcher studies the DNA sequence of a specific gene in a genome, they actually need to make many copies of that gene in order to study it. This technique, polymerase chain reaction, which is commonly abbreviated as just PCR, is used to rapidly isolate and amplify a specific sequence of DNA. Now, the term "amplify" refers to the process of making more copies of that DNA.

This is somewhat similar to DNA cloning because DNA cloning is also going to make more copies of the DNA. However, unlike DNA cloning, which uses living cells to make more copies of the DNA, polymerase chain reaction or PCR takes place in a test tube. Of course, the test tube does not include living cells. We'll be able to talk more about the process of polymerase chain reaction and PCR as we continue to move forward in our course. But for now, this concludes our brief introduction to polymerase chain reaction or PCR, and I'll see you all in our next video.

Why do we use PCR?

So now that we've briefly introduced polymerase chain reaction or PCR in our previous lesson video, it's fair to ask why do we use PCR? Well, it turns out that scientists will use PCR for many, many different reasons. And that's because PCR is a quick and efficient process for generating many identical copies of DNA in a test tube. Now DNA cloning, which occurs inside of living cells and does not occur in test tubes, is going to be more accurate, but it's actually a less efficient process. And that's because, recall, DNA cloning occurs inside of cells, and although there are fewer mutations inside of cells, it's going to take a lot longer to amplify the DNA inside of cells. And that's because a lot of times cells take 24 hours or more to be able to grow, and then after you grow the cells, you have to isolate the DNA, and that's a whole other step within itself. And so PCR is much more quick and much more efficient. And so in our example, down below, we're going to take a look at, just in general, how PCR, or what PCR can do. And so, it says here that PCR can be used to amplify the amount of DNA taken from a crime scene so that a detective can actually investigate the DNA. And so down below over here, notice we have this scientist that has a test tube here with some DNA that perhaps was found at a crime scene, but notice that there's not a whole lot of DNA within this sample. And so notice the scientist is saying, I wish I had more of this particular DNA of interest, so that he has enough DNA to be able to run tests on it. And so this is where PCR can come into play because the process of PCR, polymerase chain reaction, can amplify the amount of DNA to make many, many identical copies of the DNA as we see over here. And so the scientists are saying, wow, that amplified very, very quickly. And the process of PCR can be done in a relatively short period of time, maybe something like 2 hours, 1 and a half, 2 hours, whereas DNA cloning, again, is going to take much, much longer, which it could take well over 24 hours in many cases. And so, why do we use PCR? To make many identical copies of DNA in a test tube very, very quickly and efficiently. And so again, as we move forward in our course, we're going to continue to talk more and more about PCR and the steps of PCR. So I'll see you all in our next video.

PCR is used to _____.

• A. Amplify a single gene or small sequence of DNA.
• B. Create DNA without introns.
• C. Insert foreign DNA into a host cell where it can be replicated.
• D. Cut segments of DNA creating complimentary sticky ends.
• E. None of these.

Components of a Polymerase Chain Reaction

So before we can talk about the details of each of the steps in polymerase chain reaction or PCR, it's first helpful to talk about the components of Polymerase Chain Reaction. And so recall from our previous lesson video that PCR differs from DNA cloning in the location that the DNA is replicated. And so PCR occurs within a test tube, whereas cloning is going to occur within a cell. And so the components that go into the test tube during PCR include these components that we have listed down below. And so the components of the PCR mixture include, here is a template DNA. And so the template DNA is going to contain the sequence of interest for the study that the scientist is interested in amplifying and creating more copies of it. And so down below in this table on the left-hand side, you can see that the components of the PCR mixture in this table corresponds with the components of the PCR mixture that we have up above in the text. And so the template DNA, you can see, is right here in this image down below.

Now for the second component, there are two primers that are required, two DNA primers. And these two primers are going to be complementary to the opposite strands of DNA and are going to be oriented towards each other and serve as the starting point for the amplification. And so down below, what you can see is part here are the DNA primers, which are going to be small little DNA molecules, that are oriented opposite from one another. So notice that one goes from 5' to 3' left to right, and the other goes 5' to 3' right to left. And we'll talk more about these DNA primers and exactly how they're used as we move forward in our course.

Now the third component that's going to be needed is a thermostable DNA polymerase. And the thermostable DNA polymerase is going to be the main enzyme that's used to synthesize the sequence of interest to amplify and make more copies of the DNA of interest. And so down below here in the third component, you can see that we need a thermostable DNA polymerase, which is commonly going to be a DNA polymerase called Taq polymerase, which we'll get to talk more about Taq polymerase as we move forward in our course.

Now the fourth and final component here that's going to be needed, in the PCR mixture is, here, which is going to include all 4 deoxyribonucleotides or all 4 DNA nucleotides that are used to synthesize DNA. And so down below here in this image, you can see that it is going to include all 4 of those DNA nucleotides, which you can see over here, which include, these nucleotides that have the nitrogenous bases t, a, c, and g. And so these are the main components that are needed for a PCR mixture.

Now over here on the right-hand side, what we're showing you is a little glimpse of the process of amplifying a gene with PCR. And so, of course, you're going to start off with your template DNA, which we have here in this first column. And it turns out that PCR, polymerase chain reaction, actually occurs in a series of cycles. And so, the first cycle you can see is going to amplify the DNA. The second cycle will amplify the DNA even more, and the third cycle will amplify the DNA even more. And continuous cycles will amplify the DNA more and more and more. And so there's actually a PCR formula that can be used to determine the number of new copies of template DNA that are made at each cycle. And the formula is relatively simple. It's just 2n where n is going to act as an exponent here. And n is just a variable that represents the number of PCR cycles. And so for example, if we wanted to calculate the number of new copies of template DNA in the first cycle of PCR, all we need to do is take 21 since one is representing cycle number 1. And so 21 is equal to 2. And so notice that after the first cycle, there are 2 new copies of DNA. Now after the second cycle, if we wanted to calculate how many new copies of template DNA there would be, you just take 22. And 22 is equal to 4. And so you can see that there are 4 new copies of DNA after cycle number 2 of PCR. And the same goes for cycle number 3. If we want to calculate the number of new copies of template DNA, you just take 2 and raise it to the power of 3, which is the number of cycles, cycle 3. And 23 is equal to 8. And so you can see that there are 8 copies of DNA, after cycle number 3. And so, we'll be able to get some practice applying these concepts that we've learned as we move forward in our course. And then later, we'll get to talk about the actual steps that are involved in polymerase chain reaction or PCR. So I'll see you all in our next video.

PCR requires all of the following EXCEPT:

• A. Primers.
• B. DNA ligase.
• C. DNA polymerase.
• D. DNA of interest.
• E. Deoxyrobinucleotides.

The polymerase chain reaction:

• A. Doubles the template sequence of DNA with each cycle.
• B. Makes a single copy of the template sequence of DNA with each cycle.
• C. Is used to determine the sequence of a region of DNA.
• D. All are correct.

Here’s what students ask on this topic:

What is Polymerase Chain Reaction (PCR) and how does it differ from DNA cloning?

Polymerase Chain Reaction (PCR) is a technique used to rapidly amplify specific DNA sequences in a test tube. Unlike DNA cloning, which occurs in living cells, PCR does not require living cells and is performed entirely in vitro. PCR is highly efficient and can generate many identical copies of DNA within 1.5 to 2 hours, whereas DNA cloning is more accurate but slower, often taking over 24 hours. The key components of PCR include template DNA, two complementary primers, thermostable DNA polymerase (commonly Taq polymerase), and all four deoxyribonucleotides.

Why is PCR considered more efficient than DNA cloning?

PCR is considered more efficient than DNA cloning because it can rapidly generate many identical copies of DNA in a test tube within a short period, typically 1.5 to 2 hours. In contrast, DNA cloning involves growing cells, which can take 24 hours or more, followed by isolating the DNA, adding additional time. PCR's efficiency makes it ideal for applications requiring quick DNA amplification, such as forensic analysis or genetic research.

What are the main components required for a PCR reaction?

The main components required for a PCR reaction include: 1) Template DNA, which contains the sequence of interest; 2) Two DNA primers, complementary to the opposite strands of the DNA and oriented towards each other; 3) Thermostable DNA polymerase, commonly Taq polymerase, which synthesizes the DNA; and 4) All four deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP), which are the building blocks for new DNA strands.

How is the number of DNA copies calculated in PCR cycles?

The number of DNA copies in PCR cycles is calculated using the formula 2ⁿ, where n represents the number of cycles. For example, after the first cycle, there are 2¹ = 2 new copies of DNA. After the second cycle, there are 2² = 4 new copies, and after the third cycle, there are 2³ = 8 new copies. This exponential amplification allows for the rapid generation of large quantities of DNA.

What is the role of Taq polymerase in PCR?

Taq polymerase is a thermostable DNA polymerase used in PCR to synthesize new DNA strands. It is derived from the thermophilic bacterium Thermus aquaticus, allowing it to withstand the high temperatures required for DNA denaturation during PCR. Taq polymerase extends the DNA primers by adding nucleotides to synthesize the complementary DNA strand, enabling the amplification of the target DNA sequence.