28.8 The Polymerase Chain Reaction
3 min read•may 7, 2024
is a powerful technique that amplifies specific DNA sequences. It uses repeated cycles of heating and cooling to separate DNA strands, allow to bind, and extend new DNA strands. This process can create millions of copies from a single DNA molecule.
, a heat-stable enzyme, is key to PCR's success. It survives high temperatures and efficiently synthesizes new DNA strands. While PCR can theoretically double DNA amounts each cycle, real-world factors like enzyme efficiency impact actual rates.
The Polymerase Chain Reaction (PCR)
Steps of PCR process
Top images from around the web for Steps of PCR process
Polymerase chain reaction - Wikipedia View original
Is this image relevant?
File:PCR Steps.JPG - Wikimedia Commons View original
Is this image relevant?
10.1 Cloning and Genetic Engineering – Concepts of Biology-1st Canadian Edition View original
Is this image relevant?
Polymerase chain reaction - Wikipedia View original
Is this image relevant?
File:PCR Steps.JPG - Wikimedia Commons View original
Is this image relevant?
1 of 3
Top images from around the web for Steps of PCR process
Polymerase chain reaction - Wikipedia View original
Is this image relevant?
File:PCR Steps.JPG - Wikimedia Commons View original
Is this image relevant?
10.1 Cloning and Genetic Engineering – Concepts of Biology-1st Canadian Edition View original
Is this image relevant?
Polymerase chain reaction - Wikipedia View original
Is this image relevant?
File:PCR Steps.JPG - Wikimedia Commons View original
Is this image relevant?
1 of 3
-
- Involves heating the DNA sample to 94-96°C
- Disrupts hydrogen bonds between complementary bases (adenine-thymine, guanine-cytosine)
- Separates into single strands
- Allows primers to bind to in the next step ()
- Temperature must exceed the of the DNA
- Annealing
- Achieved by lowering the temperature to 50-65°C
- Enables primers to bind to their complementary sequences on single-stranded DNA
- Primers are short, synthetic oligonucleotides that define the starting point of DNA synthesis (forward and )
- Two primers are used, one for each strand of the DNA
-
- Occurs by raising the temperature to 72°C, the optimal temperature for Taq activity
- Taq DNA polymerase synthesizes new DNA strands complementary to the single-stranded templates
- Polymerase extends the primers by adding () to the 3' end of the primer
- Doubles the amount of DNA in the reaction ()
- This step mimics in living cells
Role of Taq DNA polymerase
- isolated from the bacterium
- Stable at high temperatures up to 94°C and has an optimal activity temperature of 72°C
- Essential for the extension step of PCR, where it synthesizes new DNA strands
- Thermostability allows it to remain active throughout multiple PCR cycles despite high denaturation temperatures
- High processivity enables synthesis of long stretches of DNA without dissociating from the template
- Use of Taq polymerase eliminates the need to add fresh polymerase after each denaturation step, making PCR more efficient and automated
Amplification factors in PCR
- Theoretical amplification factor
- Under ideal conditions, the amount of DNA doubles with each PCR cycle
- Can be calculated using the formula: , where n is the number of cycles
- After 10 cycles, the theoretical amplification factor would be
- Practical amplification factor
- In practice, PCR efficiency is not 100% due to factors such as substrate limitation, enzyme inhibition, and product accumulation
- Lower than the theoretical value
- More realistic formula for practical amplification factor is , where E is the efficiency of the reaction (typically 0.7-0.8) and n is the number of cycles
- With an efficiency of 0.75 and 10 cycles, the practical amplification factor would be
PCR Process and Equipment
- : The repeated heating and cooling of the reaction mixture for DNA melting and enzymatic replication
- Amplification: The process of increasing the number of copies of a specific DNA sequence
- : Automated device that precisely controls temperature changes for thermal cycling in PCR
Key Terms to Review (24)
Amplification: Amplification is the process of exponentially increasing the number of copies of a specific DNA sequence through repeated cycles of replication. This is a fundamental concept in the Polymerase Chain Reaction (PCR), a widely used technique in molecular biology and genetics.
Annealing: Annealing is a process in which a material, often a metal or polymer, is heated to a specific temperature, held at that temperature for a period of time, and then slowly cooled. This process is used to modify the physical and chemical properties of the material, such as increasing its strength, ductility, and resistance to deformation.
Denaturation: Denaturation is a process that occurs when the native structure of a protein or nucleic acid is disrupted, leading to the loss of its biological function. This structural change can be induced by various factors, such as temperature, pH, or the presence of certain chemicals.
DNA Polymerase: DNA polymerase is a crucial enzyme responsible for the replication and repair of DNA molecules. It plays a vital role in ensuring the accurate duplication of genetic information during cell division, as well as the maintenance of genomic integrity through DNA repair processes.
DNA Replication: DNA replication is the process by which a double-stranded DNA molecule is duplicated to produce two identical copies. It is a fundamental biological process that ensures the accurate transmission of genetic information from one generation to the next.
DNTPs: dNTPs, or deoxyribonucleotide triphosphates, are the building blocks of DNA. They are the four nucleotides (adenine, guanine, cytosine, and thymine) that are used by DNA polymerase to replicate and synthesize new DNA strands during processes like DNA sequencing and the Polymerase Chain Reaction (PCR).
Double-Stranded DNA: Double-stranded DNA (dsDNA) is the typical structure of DNA, where two complementary strands of nucleic acids are joined together to form a double helix. This structure is essential for the storage and transmission of genetic information in living organisms.
Exponential Amplification: Exponential amplification refers to the rapid and geometric increase in the number of copies of a specific DNA sequence during the Polymerase Chain Reaction (PCR) process. This term describes the exponential nature of the DNA replication that occurs in each cycle of the PCR, allowing for the generation of millions or even billions of copies from a small initial amount of DNA.
Extension: In the context of the Polymerase Chain Reaction (PCR), extension refers to the process of DNA synthesis where the DNA polymerase enzyme adds complementary nucleotides to the growing DNA strand, elongating the newly synthesized DNA molecule.
Forward Primers: Forward primers are short, synthetic DNA sequences used in the Polymerase Chain Reaction (PCR) to initiate the amplification of a target DNA sequence. They are designed to bind to the 3' end of the sense strand of the target DNA, providing a starting point for DNA synthesis by a DNA polymerase enzyme.
Melting Temperature: Melting temperature, also known as the melting point, is the temperature at which a solid substance transitions into a liquid state. This phase change occurs when the intermolecular forces that hold the solid structure together are overcome by the increased kinetic energy of the molecules, allowing them to break free and move more freely as a liquid.
Nucleotides: Nucleotides are the basic structural units of nucleic acids, such as DNA and RNA. They consist of a nitrogenous base, a five-carbon sugar, and one to three phosphate groups. Nucleotides play crucial roles in various biological processes, including energy transfer, cell signaling, and the genetic storage and transmission of information.
PCR: PCR, or Polymerase Chain Reaction, is a powerful technique used to amplify specific DNA sequences from a small amount of starting material. It is a fundamental tool in molecular biology, genetics, and biotechnology, allowing researchers to generate millions or billions of copies of a target DNA fragment for further analysis and applications.
Polymerase Chain Reaction: The Polymerase Chain Reaction (PCR) is a powerful molecular biology technique used to rapidly amplify and make millions of copies of a specific DNA sequence from a small amount of starting material. It is a fundamental tool in various fields, including genetics, forensics, and diagnostics.
Polymerase chain reaction (PCR): PCR is a laboratory technique used to amplify specific DNA sequences, making millions of copies from a small initial sample. It involves repeated cycles of temperature changes that facilitate the replication process.
Polymerase Chain Reaction Machine: The polymerase chain reaction (PCR) machine, also known as a thermal cycler, is a laboratory instrument used to amplify DNA sequences. It is a crucial tool in molecular biology, genetics, and biotechnology, enabling the rapid and exponential replication of specific DNA fragments from a small initial sample.
Primers: Primers are short, single-stranded DNA sequences that serve as starting points for DNA synthesis in various molecular biology techniques, including the Polymerase Chain Reaction (PCR). They are essential components that initiate the amplification of specific DNA sequences during the PCR process.
Reverse Primers: Reverse primers are short, synthetic DNA sequences used in the Polymerase Chain Reaction (PCR) process to initiate the replication of a specific target DNA sequence in the opposite direction. They work in conjunction with forward primers to amplify a desired genetic region.
Single-Stranded DNA: Single-stranded DNA (ssDNA) refers to a DNA molecule that consists of a single polynucleotide chain, rather than the typical double-stranded DNA (dsDNA) structure. This unique structure of ssDNA is crucial in the context of the Polymerase Chain Reaction (PCR), a widely used technique in molecular biology and genetics.
Taq Polymerase: Taq polymerase is a thermostable DNA polymerase enzyme derived from the thermophilic bacterium Thermus aquaticus. It is a crucial component in the Polymerase Chain Reaction (PCR) process, which is a powerful technique used to amplify specific DNA sequences exponentially.
Template DNA: Template DNA refers to the DNA strand that serves as the pattern or template for the synthesis of a complementary strand during DNA replication or transcription. It is the original DNA sequence that is used as a guide to create new DNA or RNA molecules.
Thermal Cycling: Thermal cycling is a fundamental process in the Polymerase Chain Reaction (PCR) technique, which is used to amplify specific DNA sequences. It involves the repetition of a series of temperature changes to facilitate the different stages of DNA replication, enabling the exponential multiplication of target DNA fragments.
Thermostable DNA Polymerase: A thermostable DNA polymerase is an enzyme that can withstand high temperatures and is used in the Polymerase Chain Reaction (PCR) to amplify specific DNA sequences. These enzymes are derived from thermophilic organisms, which thrive in high-temperature environments, and are crucial for the success of the PCR technique.
Thermus aquaticus: Thermus aquaticus is a species of bacterium that is notable for its ability to survive and thrive in extremely hot environments. This thermophilic bacterium is a key component in the Polymerase Chain Reaction (PCR), a widely used technique in molecular biology and genetics.