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.
노트:
- 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 박테리아.
Simple and fast
- PCR reactions use heat-resistant Taq DNA polymerase.
- Once the reaction mixture is prepared, 변성, 가열 냉각, and extension reactions are carried out on a DNA amplification liquid and a water bath, typically completing the amplification reaction in 2 에게 4 시간.
- 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, 체액, 담, 머리카락, 세포, 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.