Development of PCR tech
- Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments.
- It can be considered a special type of DNA replication that occurs outside of living organisms.
- DNA polymerase I was first discovered in 1955.
- The Klenow fragment of E. coli, discovered in the early 1970s by Dr. H. Klenow, has experimental value and practicality.
- Međutim, this enzyme is not heat-resistant and denatures at high temperatures, making it unsuitable for use in PCR that requires high-temperature denaturation.
- The enzyme commonly used today, referred to as Taq polymerase, was isolated from the bacterium Thermus aquaticus in 1976.
- Its characteristic of heat resistance makes it an ideal enzyme, but its widespread use came after the 1980s.
- The original concept of PCR, similar to gene repair replication, was proposed by Dr. Kjell Kleppe in 1971.
- He published the first experiment of pure and transient gene replication (similar to the first two cycles of PCR).
- The PCR developed today was pioneered by Dr. Kary B. Mullis in 1983, when Mullis worked for the company PE, giving the company a special position in the PCR field.
- Mullis and Saiki formally published the first related paper in 1985.
- Since then, the application of PCR has expanded rapidly, and the quality of related papers has surpassed many other research methods.
- Subsequently, PCR technology has been widely used in biological research and clinical applications, becoming a crucial technique in molecular biology research.
- Mullis was awarded the Nobel Prize in Chemistry in 1993 for his contribution.
The principle of PCR
The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity depends on the oligonucleotide primers that are complementary to the target sequence at both ends. PCR consists of three basic reaction steps: denaturation, annealing, and extension:
- Denaturation of template DNA: After heating the template DNA to around 93°C for a certain period of time, the double-stranded DNA of the template DNA or the double-stranded DNA formed by PCR amplification is dissociated, making it single-stranded so that it can bind to the primers and prepare for the next round of reaction.
- Annealing (reassociation) of template DNA and primers: After the template DNA is denatured into single strands, the temperature is reduced to around 55°C, and the complementary sequences of the primers and the single-stranded template DNA pair up and bind.
- Extension of primers: With the action of Taq DNA polymerase, using dNTPs as reaction substrates and the target sequence as the template, according to the principle of complementary base pairing and semi-conservative replication, a new semi-conservative replication chain complementary to the template DNA chain is synthesized. This chain can be amplified into millions of copies of the target gene within 2 to 3 hours by repeating the denaturation, annealing, and extension processes.
PCR reaction system and reaction conditions Standard PCR reaction system
| Component | Volume/Concentration |
|---|---|
| 10x amplification buffer | 10μl |
| 4 types of dNTP mixture | 200μl |
| Primers | 10-100μl |
| Template DNA | 0.1-2μg |
| Taq DNA polymerase | 2.5μl |
| Mg2+ | 1.5mmol/L |
| Double or triple distilled water | 100μl |
The five elements of PCR reaction:
- Primers (PCR primers are DNA fragments, while primers for cellular DNA replication are RNA chains)
- Enzim
- dNTPs
- Template
- Buffer solution (which requires Mg2+)
The standard PCR process consists of three steps:
- Denaturation of DNA (90°C-96°C): Under the influence of heat, the hydrogen bonds in the double-stranded DNA template break, forming single-stranded DNA.
- Annealing (25°C-65°C): The system temperature decreases, allowing the primers to bind to the DNA template, forming local double-stranded regions.
- Extension (70°C-75°C): With the action of Taq polymerase (optimal activity around 72°C), using dNTPs as substrates, extension occurs from the 5′ end to the 3′ end of the primer, synthesizing a DNA strand complementary to the template.
Notes:
- Each cycle undergoes denaturation, annealing, and extension, doubling the amount of DNA.
- Some PCR protocols, due to the short amplification region, can complete replication even if Taq polymerase activity is not optimal.
- In these cases, a two-step method can be used, where annealing and extension occur simultaneously at a temperature between 60°C and 65°C, reducing the number of temperature cycles and thus improving reaction speed.
PCR reaction characteristics
Strong specificity
The specificity of the PCR reaction is determined by:
- Specific and correct binding of primers to the template DNA.
- Base pairing principles.
- Faithfulness of Taq DNA polymerase synthesis reaction.
- Specificity and conservation of the target gene.
Notes:
- Correct binding of primers to the template is crucial.
- The binding of primers to the template and the extension of primer chains follow the principles of base pairing.
- The fidelity of the polymerase synthesis reaction and the heat resistance of Taq DNA polymerase allow the annealing (reassociation) of the template and primers to occur at higher temperatures, greatly increasing the specificity of binding and maintaining a high level of accuracy for the amplified target gene segment.
- Furthermore, by selecting target gene regions with high specificity and conservation, the level of specificity is further increased.
High sensitivity
- The amount of PCR product increases exponentially, enabling the amplification of starting template amounts from picograms (pg=10^-12) to micrograms (μg=10^-6).
- It can detect a target cell from one million cells.
- In virus detection, PCR sensitivity can reach 3 RFU (recombinant forming units), while in bacteriology, the minimum detection rate is 3 bacteria.
Simple and fast
- PCR reactions use heat-resistant Taq DNA polymerase.
- Once the reaction mixture is prepared, denaturation, annealing, and extension reactions are carried out on a DNA amplification liquid and a water bath, typically completing the amplification reaction in 2 to 4 sati.
- Amplification products are usually analyzed by electrophoresis, without the necessity of using isotopes, thus minimizing radioactive contamination and facilitating dissemination.
Low purity requirements for specimens
- There is no need to isolate viruses or bacteria or culture cells; crude DNA and RNA can be used as amplification templates.
- Clinical specimens such as blood, body fluids, sputum, hair, cells, and live tissue can be directly used for DNA amplification detection.
PCR troubleshooting
False negatives
The absence of amplification bands in PCR reactions is often caused by key factors:
Template Nucleic Acid Preparation
- Contaminants such as proteins in the template, the presence of Taq polymerase inhibitors, incomplete removal of proteins (especially histones) from chromosomal DNA, excessive loss during template extraction, or phenol inhalation can contribute to the issue.
- Incomplete denaturation of template nucleic acids may also play a role.
- When enzyme and primer qualities are good, and amplification bands don’t appear, it’s likely due to sample digestion issues or faults in the template nucleic acid extraction process.
- Effective and stable digestion solutions should be prepared, and the procedure should remain fixed.
Enzyme Inactivation
This may necessitate replacing the enzyme with a new one or using a combination of old and new enzymes to analyze whether enzyme activity loss or insufficiency leads to false negatives. Sometimes, forgetting to add Taq polymerase or ethidium bromide can also cause issues.
Primer Issues
- Quality and concentration of primers, symmetry of primer concentrations, and batch variations in primer synthesis quality can impact PCR outcomes.
- Solutions include selecting reputable primer synthesis units, confirming primer concentrations through agarose gel electrophoresis, ensuring similar band intensities for both primers, storing primers at high concentrations in small aliquots to prevent degradation, and ensuring rational primer design to prevent issues like primer dimer formation.
Mg2+ Concentration
Mg2+ ion concentration significantly affects PCR amplification efficiency. Excessive or inadequate concentrations can affect specificity or yield, leading to failed amplification.
Reaction Volume Changes
PCR volumes typically range from 20μl to 100μl, depending on the purpose. Changing volumes should be carefully adjusted to prevent failures.
Physical Factors
Denaturation is crucial, and inadequate denaturation or annealing temperatures can cause false negatives. Using standard thermometers to check thermocycler or water bath temperatures can help prevent failures.
Target Sequence Variations
Mutations or deletions in the target sequence can affect primer-template binding, leading to failed amplification.
False Positives
The observed PCR amplification bands match the target sequence bands, sometimes appearing more uniform and brighter. Possible causes:
Inappropriate Primer Design:
Selection of amplification sequences with homology to non-target sequences can result in non-specific amplification products during PCR. Short target sequences or primers can lead to false positives. Redesigning primers is necessary.
Cross-contamination of Target Sequences or Amplification Products:
Contamination Causes:
- Contamination of the entire genome or large segments can lead to false positives.
- Contamination of small nucleic acid fragments in the air can also cause false positives.
How to handle this situation
1. Strict Laboratory Practices:
- Handle samples with care to prevent the inhalation of target sequences into pipettes or splashing outside centrifuge tubes.
- Autoclave all reagents and equipment, except for enzymes and materials intolerant to high temperatures.
- Use disposable centrifuge tubes and pipette tips to minimize contamination.
2. UV Exposure:
- Expose reaction tubes and reagents to ultraviolet light before sample addition to destroy existing nucleic acids.
- Primer Design Consideration:
- Design primers carefully to minimize the potential for cross-reactivity with non-target sequences.
- Nested PCR Methods:
- Implement nested PCR methods to mitigate or eliminate false positives caused by contamination of small nucleic acid fragments in the air.
Appearance of non-specific amplification bands
- After PCR amplification, bands appear that are inconsistent in size with the expected ones, either larger or smaller, or both specific and non-specific bands appear simultaneously.
- The reasons for the appearance of non-specific bands are:
- Incomplete complementarity between primers and target sequences, or primer dimer formation.
- Excessive Mg2+ ion concentration, too low annealing temperature, and excessive PCR cycle number.
- Quality and quantity of the enzyme, with enzymes from some sources prone to non-specific bands, while others are not.
- Excessive enzyme quantity can sometimes lead to non-specific amplification.
- Countermeasures include:
- Redesigning primers if necessary.
- Reducing enzyme quantity or switching to enzymes from different sources.
- Lowering primer concentration, increasing template concentration appropriately, and reducing the number of cycles.
- Raising the annealing temperature appropriately or adopting a two-temperature point method (denaturation at 93°C, annealing and extension at around 65°C).
Appearance of smearing or streaking bands
PCR amplification sometimes results in smearing or streaking bands.
- This is often caused by excessive enzyme quantity or poor enzyme quality, high dNTP concentration, excessive Mg2+ concentration, low annealing temperature, or excessive cycle number.
- Countermeasures include:
- Reducing enzyme quantity or switching to enzymes from different sources.
- Decreasing dNTP concentration.
- Appropriately lowering Mg2+ concentration.
- Increasing template quantity.
- Reducing cycle number.
