PCR Cycling Process – An Overview of the 3 Stages

PCR (Polymerase Chain Reaction) amplifies DNA and involves a series of temperature cycles critical for amplifying the DNA target. The cycling process is divided into three main stages: denaturation, annealing, and extension.

These three stages are repeated for 20-40 cycles, doubling the targeted DNA amount. This PCR cycling process is a highly efficient way to amplify specific DNA sequences, which makes it an essential technique in molecular biology and genetic research.

3 Stages of the PCR Cycling Process

Polymerase Chain Reaction (PCR) is a powerful molecular biology technique used to amplify specific regions of DNA. The PCR cycle consists of three main stages: initial denaturation, cycling, and repeat. Each stage plays a crucial role in successfully amplifying the target DNA sequence.

• Initial Denaturation
  This stage is typically conducted at a high temperature (around 95-98°C) for 1-5 minutes. The high temperature causes  the hydrogen bonds between the two strands of the DNA double helix to break, separating the strands and creating single-stranded DNA templates. This stage is essential to initiate the amplification of the targeted DNA sequence.
• Cycling
  The second stage of the PCR cycling process is the cycling stage and consists of three sub-steps:
  denaturation, annealing, and extension. Each sub-step is performed at a specific temperature and for a defined period.

The temperature and time for each sub-step are preset, based on the specific DNA target, the primers used, and the type of polymerase enzyme. Below are the steps described in detail:

1. Denaturation
   During this step, the temperature increases to around 95-98°C for 15-30 seconds. The high temperature causes the double-stranded DNA template to denature, resulting in two single-stranded DNA templates.
2. Annealing
   This step involves lowering the temperature to around 50-56°C for specific amplification and 36°C for aspecific amplification. At this temperature, the primers bind to the single-stranded DNA templates to provide a starting point for the polymerase enzyme.
3. Extension
   The temperature is raised to around 72-75°C for 15-30 seconds during this step. The polymerase enzyme extends the primers by adding nucleotides to the 3' end of the DNA template, creating a new double-stranded DNA molecule.

• Repeat

The repeat stage is the third stage in the PCR cycling process. The cycling stage is repeated for 20-40 cycles, with each cycle doubling the DNA target amount. The final number of cycles is optimized to ensure efficient amplification of the target DNA sequence without generating nonspecific amplification products.

PCR temperature cycle

PCR (Polymerase Chain Reaction) amplifies the specific DNA regions. The three main temperature stages are denaturation, annealing, and extension, which are already discussed in detail above.

The cycle then repeats, starting with the denaturation stage. The number of cycles can range from 20 to 40, depending on the DNA concentration required for downstream applications. Temperature and time parameters must be optimized for each stage of the PCR cycle based on the specific DNA target, the primers used, and the type of polymerase enzyme.

That makes the PCR temperature cycle a critical component of PCR, with each stage requiring specific temperature and time parameters to ensure efficient amplification of the targeted DNA sequence. 

Denaturation in PCR

Denaturation is the first stage of the Polymerase Chain Reaction (PCR) process. I t involves separating the two strands of the DNA double helix to create single-stranded DNA templates.

During the denaturation stage, the temperature is typically set to 94-98°C for 20-30 seconds. The hydrogen bonds joining the strands of the DNA double helix together are broken, causing the double-stranded DNA molecule to unwind and separate into two single-stranded DNA templates. This is a crucial step in the PCR process that involves exposing the template DNA and allowing the primers to bind to the template during the annealing stage.

The stage lasts for a short period of time, and the temperature is optimally maintained to ensure that the double-stranded DNA is completely denatured. The temperature and time parameters for the denaturation stage can vary depending on the length and GC content of the DNA target, the type of polymerase used, and the type of PCR instrument.

In addition to temperature and time, the quality of the DNA template, the concentration of PCR components, and the pH of the reaction mixture can also affect the efficiency and specificity of the denaturation stage.

Optimizing Denaturation PCR

It’s important to optimize the denaturation step in PCR to ensure the amplification of specific regions of DNA. Denaturation requires separating two DNA strands and creating single-stranded DNA templates that can be utilized for amplification. Here are some tips on optimizing the denaturation stage of PCR:

• Temperature and Duration
  The temperature and duration of the denaturation stage are critical for the success of the PCR reaction. The optimal temperature is usually in the range of 94-98°C, and the duration of denaturation is typically 20-30 seconds. Both parameters can vary depending on the length and GC content of the DNA target and the type of polymerase used.
• DNA Template Quality
  The DNA template’s quality is an important factor affecting the denaturation stage's efficiency. Poor-quality DNA can result in incomplete denaturation, leading to lower efficiency of the PCR reaction. In contrast, high-quality DNA is free from impurities and facilitates a successful denaturation step.
• Concentration of PCR Components
  The concentration of PCR components can affect the efficiency of the denaturation stage. The optimal concentration of the DNA template, primers, and polymerase should be determined to achieve maximum efficiency.
• pH of the Reaction Mixture
  The reaction mixture's pH can affect the denaturation stage's efficiency. That’s why it’s essential to optimize the pH of the reaction mixture to ensure that the reaction conditions are optimal for denaturation.
• PCR Instrument
  The type of PCR instrument used can affect the efficiency of the denaturation stage. Different instruments have different heating and cooling rates that affect the denaturation step. That’s why it’s suggested to determine the optimal denaturation temperature and time based on the instrument used.

Primer annealing PCR

The second stage is primer annealing, where the temperature is lowered to around 50-65°C for 20-30 seconds to let the primers bind to the complementary regions of the single-stranded DNA templates. Primers are short, synthetic DNA sequences designed to hybridize specifically to the target DNA region. The annealing temperature is critical for successful PCR amplification and must be optimized to prevent the primers from binding to non-specific regions.

Optimizing Annealing PCR

You can optimize the annealing step in the PCR to achieve high PCR amplification efficiency and specificity. Below are some tips and considerations to optimize the annealing stage in PCR:

• Temperature
  The annealing temperature should be optimized based on the melting temperature (T_m) of the primers used. The T_m of the primers is the temperature at which half of the primers are hybridized to the DNA template while the other half isn’t. Typically, the annealing temperature is set at 3-5°C below the T_m of the primers.
• Time
  The annealing time should be long enough to let primers hybridize into the DNA template. Typically, annealing times range from 15-30 seconds.
• Magnesium Ion Concentration
  Magnesium ions are critical for PCR amplification as they activate the DNA polymerase. The optimal magnesium ion concentration should be experimentally optimized for each primer pair, as too little can reduce amplification efficiency, while too much can cause non-specific amplification.
• Annealing Buffer Composition
  The composition of the annealing buffer can also affect the efficiency of the annealing step. The buffer should be optimized for each primer pair to ensure optimal annealing temperature and time.
• Primer Design
  The design of the primer is critical for achieving optimal annealing. They should be designed to have similar melting temperatures and GC content to avoid template regions that are likely to form secondary structures. Guanine-cytosine content is the amount of nitrogenous bases in DNA or RNA molecules that are guanine (G) or cytosine (C). The higher the GC content of DNA, the higher its melting temperature.    
• Template Quality
  The template DNA's quality can affect the annealing step's efficiency. The template should be high quality, free from impurities, and with good concentration.
• Annealing Temperature Gradient
  Performing a temperature gradient PCR can effectively optimize the annealing temperature. It involves running the PCR reaction simultaneously at various annealing temperatures to determine the optimal annealing temperature.

Primer extension PCR

Primer extension typically occurs at a temperature ranging from 68-72°C. The Taq polymerase enzyme extends the primers by adding nucleotides to the 3' end of the primers, resulting in the synthesis of a new DNA strand. The extension time depends on the target sequence’s length and is typically 30 seconds to a few minutes.

The three steps are repeated for multiple cycles, with each cycle doubling the amount of DNA in the sample. The number of cycles is determined by the amount of target DNA in the initial sample and the sensitivity required for the experiment.

PCR Extension Considerations

PCR extension is an essential step in the PCR process, during which the DNA polymerase enzyme extends the primers by adding nucleotides to the 3' end of the primers, resulting in the synthesis of a new DNA strand. Here are some tips and considerations for optimizing this stage of PCR:

• Optimize the Extension Time and Temperature
  The extension time and temperature depend on the length of the target sequence and the DNA polymerase used. Longer target sequences require greater extension times, with some DNA polymerases working optimally at higher temperatures. 
  
  As a result, it is essential to determine the optimal extension time and temperature for the PCR reaction to ensure the amplification of the target sequence.
• Consider the Mg2+ Concentration
  The Mg2+ concentration in the PCR reaction can affect the extension step’s efficiency. Too little Mg2+ can result in incomplete extension, while too much Mg2+ can inhibit the PCR reaction. As a result, it is important to optimize the Mg2+ concentration for your specific PCR reaction.
• Avoid Template Degradation
  If the reaction is carried out for too long or at too high a temperature, it can result in template degradation. The extension time should be as short as possible and temperature as low as possible to avoid prolonged exposure to high temperatures to avoid template degradation.
• Consider Using Proofreading Polymerase
  Some DNA polymerases have a proofreading activity to rectify the errors introduced during DNA synthesis. A proofreading polymerase can increase the PCR reaction’s accuracy, which may be important for sequencing.
• Use a High-Quality DNA Template
  The DNA template quality can affect the PCR reaction's efficiency and specificity. Using a high-quality DNA template free of impurities, such as protein or salts, is important to not inhibit the PCR reaction.
• Consider the Primer Design
  The primer design can also affect the efficiency and specificity of the PCR reaction. For example, primers with high GC content can require higher annealing temperatures, while primers with low GC content can require lower annealing temperatures. That’s why it is important to carefully design the primers to ensure efficient and specific amplification of the target sequence.

Determining the number of PCR cycles

A crucial step in the polymerase chain reaction (PCR) process is determining the number of PCR cycles required to obtain the desired concentration of the target DNA sequence. 

Generally, the number of cycles required for PCR amplification depends on the initial amount of target DNA, the sensitivity of the detection method, and the desired level of amplification. PCR aims to amplify the target DNA to a level where it can be easily detected or further analyzed.

However, it is essential to remember that excessive amplification can lead to non-specific amplification or the formation of unwanted by-products, which can compromise the PCR product’s quality. Therefore, PCR should be performed with the minimum number of cycles required for the desired level of amplification.

It is important to consider several factors, such as the length of the target DNA sequence, the efficiency of the primers, the type of DNA polymerase used, and the thermocycling conditions to determine the optimal number of PCR cycles. Typically, 20-30 cycles are sufficient for most PCR applications. The optimal number of cycles may vary depending on the specific application.

One way to determine the optimal number of PCR cycles is to perform a pilot experiment with a range of cycle numbers and compare the resulting amplification products. Analyzing the PCR products on an agarose gel helps determine the optimal cycle number that provides sufficient amplification without non-specific products.

The number of PCR cycles can affect the PCR product’s accuracy. In general, a higher number of cycles can lead to the accumulation of errors or mutations in the PCR product. The number of cycles used to reach the desired level of amplification should be minimized.

The Final PCR Primer Extension Step

The final PCR primer extension step is critical in the polymerase chain reaction (PCR) process. Once the extension step is complete, the PCR reaction undergoes a final cooling step to allow the DNA strands to anneal and the reaction to halt. The final PCR product can then be analyzed using various techniques, such as gel electrophoresis or DNA sequencing.

Key PCR Assay Components to Consider

PCR assay components include a template DNA, primers, Taq polymerase or another DNA polymerase, dNTPs (deoxynucleoside triphosphates), buffer, and magnesium ions. 

Primers are short single-stranded DNA sequences that flank the target DNA sequence. The DNA polymerase enzyme is required to synthesize new DNA strands, while dNTPs provide the nucleotides required for DNA synthesis. 

The buffer maintains the pH and salt concentration of the reaction mixture, while magnesium ions are necessary for the activity of the DNA polymerase enzyme. All of these components are critical for the success of the PCR reaction.

DNA Polymerase

DNA polymerase is an enzyme that catalyzes  the synthesis of new DNA strands using single-stranded DNA as a template. It is a critical component of the PCR reaction as it helps amplify the target DNA sequence. 

DNA polymerases are highly specific and can only add nucleotides to the 3' end of a primer base paired with a complementary template strand. DNA polymerase is essential for the efficient and accurate amplification of the target DNA sequence in PCR reactions.

PCR Workstations

PCR workstations are specialized enclosures designed to minimize contamination during the PCR process. They provide a sterile environment for preparing and handling PCR samples while preventing the introduction of airborne contaminants that can interfere with the accuracy of the results. These workstations are equipped with UV lights, HEPA filters, and negative air pressure systems to maintain a sterile environment.

Templates

Templates are the starting material for PCR amplification. DNA or RNA templates are purified from various sources such as cells, tissues, or biological fluids. Avantor Sciences offers a range of products for DNA and RNA purification, including DNA purification kits and RNA purification kits. These kits can help researchers obtain high-quality nucleic acid samples for downstream applications, such as PCR.

Learn more about PCR

You can go to the following pages to learn more about polymerase chain reaction:
What is polymerase chain reaction (PCR)?
What is a PCR workstation?
Real-time PCR (qPCR): A comprehensive overview