→ describe the steps of PCR technique ←
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Introduction and Historical Timelines
Polymerase Chain Reaction
Quantitative PCR
Digital PCR
Quantitative PCR and Digital PCR Detection Methods
PCR/qPCR/dPCR Assay Design
Sample Purification and Quality Assessment
Reverse Transcription
Assay Optimization and Validation
Data Analysis
Troubleshooting
Regulatory Agencies and Conclusions
Additional Resources
Protocols
Introduction and Historical Timelines
Polymerase Chain Reaction
Quantitative PCR
Digital PCR
Quantitative PCR and Digital PCR Detection Methods
PCR/qPCR/dPCR Assay Design
Sample Purification and Quality Assessment
Reverse Transcription
Assay Optimization and Validation
Data Analysis
Troubleshooting
Regulatory Agencies and Conclusions
Additional Resources
Protocols
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Review of PCR Reagents
Reporter: Professor Pear, thank you for taking the time to explain the forensic evidence in the case that exonerated Colonel Custard and implicated Mr. Teal in the spiral staircase with the lead pipe that killed Mr. Bones.
Professor Pear: Oh, my pleasure. Isn't it amazing how a forensic scientist can use PCR to take a small amount of DNA from a crime scene and make enough copies for analysis? PCR stands for polymerase chain reaction, and it's a laboratory procedure that can be used to create copies of DNA. There are five basic reagents, or ingredients, necessary for PCR: template DNA, PCR primers, nucleotides, Taq polymerase and PCR buffer.
Reporter: Yes, yes. Thank you for the review, Professor, but you already explained to me what reagents are necessary for PCR. Now, can you please explain how the reagents are used to make more DNA?
Professor Pear: Sorry. These reagents are mixed in a PCR tube and placed in a PCR machine. It'll soon become apparent why a machine is really important, but let me explain the steps in PCR first.
Denaturation Step
Professor Pear: Remember that PCR is kind of like artificial replication in a tube. In DNA replication, the enzyme helicase broke the hydrogen bonds between complementary base pairs.
Reporter: But, we're not adding helicase to the PCR tube, so it's not going to work!
Professor Pear: Well, yes and no. It's true that we're not adding helicase, but there's a really good reason. While they're really cool, enzymes are kind of expensive and sometimes finicky to work with. Scientists came up with a simpler, much less expensive way to break hydrogen bonds: simply heat the solution. By raising the temperature of the PCR solution to just below boiling, all the hydrogen bonds holding the complementary strands of DNA together are broken. This is known as the denaturation step in PCR.
Reporter: Oh, now I see why you can't use human polymerase. This step would kill the protein because a human protein isn't designed to function at this temperature. But, Taq polymerase works in PCR because it comes from a bacterium that normally lives in hot springs.
Professor Pear: That's exactly right.
Annealing Step
Professor Pear: If we again use DNA replication as a guide, the next step that needs to occur is that primers need to attach to the DNA template. In replication, an enzyme - RNA primase - creates the primer nucleotide-by-nucleotide on the DNA template.
In PCR, many man-made DNA primer molecules are already in the PCR tube. During the second step of PCR, called the annealing step, the primers attach, or anneal, to the DNA template. PCR requires two different primers, one that can attach to each strand of the DNA molecule.
Extension/Elongation Step
Professor Pear: Recall that PCR is only going to amplify a certain region of DNA, not the entire DNA template. The primers represent the starting point for the next step, called the extension step. During the extension, or elongation, step, Taq polymerase binds to each PCR primer and begins adding nucleotides. Note that Taq, like human DNA polymerase, can only add DNA nucleotides in one direction.
This is the reason why you need two primers that are located on either side of the region you wish to amplify. Each primer is oriented to amplify one of the complementary strands. The extension step is so named because the product of PCR continues to extend during this step.
Reporter: Wait! I don't get it. Look at the PCR products. I thought you said that this was a precise process, but, that doesn't seem very specific or precise at all. It looks like more than just the target sequences got copied.
PCR Cycling
Professor Pear: Good point. The problem is that we're not done with the PCR process yet. PCR is typically performed in cycles. That is, I've mentioned three separate steps that can produce two copies of our target DNA from a single copy. To make millions of copies, we need to perform these three steps multiple times.
Reporter: Professor Pear, thank you for taking the time to explain the forensic evidence in the case that exonerated Colonel Custard and implicated Mr. Teal in the spiral staircase with the lead pipe that killed Mr. Bones.
Professor Pear: Oh, my pleasure. Isn't it amazing how a forensic scientist can use PCR to take a small amount of DNA from a crime scene and make enough copies for analysis? PCR stands for polymerase chain reaction, and it's a laboratory procedure that can be used to create copies of DNA. There are five basic reagents, or ingredients, necessary for PCR: template DNA, PCR primers, nucleotides, Taq polymerase and PCR buffer.
Reporter: Yes, yes. Thank you for the review, Professor, but you already explained to me what reagents are necessary for PCR. Now, can you please explain how the reagents are used to make more DNA?
Professor Pear: Sorry. These reagents are mixed in a PCR tube and placed in a PCR machine. It'll soon become apparent why a machine is really important, but let me explain the steps in PCR first.
Denaturation Step
Professor Pear: Remember that PCR is kind of like artificial replication in a tube. In DNA replication, the enzyme helicase broke the hydrogen bonds between complementary base pairs.
Reporter: But, we're not adding helicase to the PCR tube, so it's not going to work!
Professor Pear: Well, yes and no. It's true that we're not adding helicase, but there's a really good reason. While they're really cool, enzymes are kind of expensive and sometimes finicky to work with. Scientists came up with a simpler, much less expensive way to break hydrogen bonds: simply heat the solution. By raising the temperature of the PCR solution to just below boiling, all the hydrogen bonds holding the complementary strands of DNA together are broken. This is known as the denaturation step in PCR.
Reporter: Oh, now I see why you can't use human polymerase. This step would kill the protein because a human protein isn't designed to function at this temperature. But, Taq polymerase works in PCR because it comes from a bacterium that normally lives in hot springs.
Professor Pear: That's exactly right.
Annealing Step
Professor Pear: If we again use DNA replication as a guide, the next step that needs to occur is that primers need to attach to the DNA template. In replication, an enzyme - RNA primase - creates the primer nucleotide-by-nucleotide on the DNA template.
In PCR, many man-made DNA primer molecules are already in the PCR tube. During the second step of PCR, called the annealing step, the primers attach, or anneal, to the DNA template. PCR requires two different primers, one that can attach to each strand of the DNA molecule.
Extension/Elongation Step
Professor Pear: Recall that PCR is only going to amplify a certain region of DNA, not the entire DNA template. The primers represent the starting point for the next step, called the extension step. During the extension, or elongation, step, Taq polymerase binds to each PCR primer and begins adding nucleotides. Note that Taq, like human DNA polymerase, can only add DNA nucleotides in one direction.
This is the reason why you need two primers that are located on either side of the region you wish to amplify. Each primer is oriented to amplify one of the complementary strands. The extension step is so named because the product of PCR continues to extend during this step.
Reporter: Wait! I don't get it. Look at the PCR products. I thought you said that this was a precise process, but, that doesn't seem very specific or precise at all. It looks like more than just the target sequences got copied.
PCR Cycling
Professor Pear: Good point. The problem is that we're not done with the PCR process yet. PCR is typically performed in cycles. That is, I've mentioned three separate steps that can produce two copies of our target DNA from a single copy. To make millions of copies, we need to perform these three steps multiple times.
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