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

Polymerase chain reaction (PCR) is a technique used to amplify DNA through a series of cycles involving denaturation, annealing, and extension. Initially, DNA strands are separated at 95°C, followed by cooling to allow primers to bind at 55-70°C. DNA polymerase then synthesizes new DNA strands at around 78°C. This process can be repeated 25-40 times, resulting in billions of copies, essential for applications in disease diagnosis and forensic analysis. Quantitative PCR (qPCR) and reverse transcription PCR (RT-PCR) are variations that quantify DNA and convert RNA to DNA, respectively.

PCR

Hi. In this video, I'm going to be talking to you about polymerase chain reaction or PCR. PCR is a method of amplifying DNA. Essentially, the basis of it is that it includes multiple cycles, usually 25 to 40, of these three steps. The first step that happens is you have a DNA you want to amplify, and you expose it to high temperatures, which separates those two strands. The DNA is double-stranded, but in order to replicate it, you have to separate them. The high temperatures break those hydrogen bonds and separate out the DNA. This usually happens at 95 degrees Celsius.

Then, after you separate the DNA, you need enzymes to be able to work, and typically, they don't work at this high temperature. So then you lower the temperature to somewhere between 55 to 70 degrees. What this does is it allows for primers to bind. What are primers, you ask? Primers are small nucleotide sequences that are complementary to the DNA you're trying to replicate. They will bind to that DNA at this lower temperature, signaling to DNA polymerase that this is the sequence you're going to replicate. Scientists design these primers so that they indicate the start site, the stop site, and where replication should occur.

The temperature is usually then raised to around 78 degrees. Essentially, DNA polymerase is added, and then it can begin the DNA replication. Once this is done for a while, you start back. So, you get multiple cycles, like I said, 25 to 40, and this allows for generating a ton of copies of DNA. Why do we do this? Well, it allows scientists to create billions of copies of DNA, which can be used in diagnosing diseases or in forensics applications, like determining who the DNA evidence at a crime scene belongs to. It is important for scientists who need to compare DNA molecules because having just one DNA molecule doesn't provide enough information for examination.

You need multiple copies of it to be able to use technology to determine its sequence and how it compares. This technique can be used to test for the presence of a specific DNA sequence. If it can be amplified, there will be tons of it; if it cannot, it is not present. There are also newer technologies that allow you to actually calculate the exact amount of DNA present in a sample. This type of analysis is called qPCR, which stands for quantitative PCR. If you start with RNA, typically, you do RT-PCR, which stands for reverse transcription PCR. This uses special techniques and dyes, but essentially follows the same process of multiple cycles, to determine how much DNA is in the sample and compare it across various samples.

This is very important. So, this is an example. We start out with some DNA fragment that we want to amplify, shown here in red. We take primers, and they bind here, signaling to DNA polymerase to come and bind. The DNA polymerase will come, bind there, and replicate. After cycle 1, you get 4 copies. After cycle 2, you get 8 copies. I didn't show cycle 3 here, but if I did, it would result in 2, 4, 6, 8, 10, 12, 14, 16 copies, and so on. As mentioned, this is performed over 25 to 40 cycles. You can imagine the number of DNA molecules you get is extremely high, and that can be very useful in a laboratory setting. That's PCR, let's now move on.

How many copies of DNA exist after 4 cycles of PCR?

• A. 4
• B. 8
• C. 16
• D. 32

Which of the following shows the correct order of steps for PCR?

• A. Melting DNA strands → Primer Binding → DNA polymerase replication
• B. Primer Binding → Melting DNA strands → DNA polymerase replication
• C. Melting DNA strands → DNA polymerase replication → Primer Binding

Here’s what students ask on this topic:

What is the purpose of Polymerase Chain Reaction (PCR)?

The purpose of Polymerase Chain Reaction (PCR) is to amplify specific DNA sequences, generating billions of copies from a small initial sample. This is crucial for various applications, including diagnosing diseases, forensic analysis, and genetic research. By producing a large quantity of DNA, scientists can analyze genetic material more effectively, compare DNA sequences, and detect the presence of specific genes or mutations. PCR is also foundational for advanced techniques like quantitative PCR (qPCR) and reverse transcription PCR (RT-PCR), which allow for DNA quantification and RNA analysis, respectively.

How does the PCR process work?

The PCR process involves three main steps: denaturation, annealing, and extension. First, the DNA sample is heated to 95°C to separate the double-stranded DNA into single strands (denaturation). Next, the temperature is lowered to 55-70°C to allow primers to bind to the target DNA sequences (annealing). Finally, the temperature is raised to around 78°C, enabling DNA polymerase to synthesize new DNA strands by adding nucleotides to the primers (extension). These steps are repeated for 25-40 cycles, exponentially amplifying the DNA.

What are the differences between qPCR and RT-PCR?

Quantitative PCR (qPCR) and Reverse Transcription PCR (RT-PCR) are variations of the standard PCR technique. qPCR, also known as real-time PCR, quantifies the amount of DNA in a sample by measuring the fluorescence emitted during the amplification process. This allows for the determination of the initial quantity of DNA. RT-PCR, on the other hand, starts with RNA instead of DNA. It involves converting RNA into complementary DNA (cDNA) using reverse transcriptase before amplifying the cDNA through standard PCR. RT-PCR is commonly used to study gene expression.

Why is Taq polymerase used in PCR?

Taq polymerase is used in PCR because it is a heat-stable enzyme derived from the thermophilic bacterium Thermus aquaticus. This enzyme can withstand the high temperatures (around 95°C) required for the denaturation step of PCR without denaturing itself. Its stability and efficiency at high temperatures make it ideal for synthesizing new DNA strands during the extension step, ensuring the PCR process can be repeated for multiple cycles without the enzyme losing its activity.

What are primers in PCR, and why are they important?

Primers are short, single-stranded nucleotide sequences that are complementary to the target DNA region to be amplified. They are crucial in PCR because they provide a starting point for DNA synthesis. During the annealing step, primers bind to their complementary sequences on the single-stranded DNA. This binding signals the DNA polymerase to start adding nucleotides at the primer site, enabling the synthesis of new DNA strands. Without primers, the DNA polymerase would not know where to begin replication.