Polymerase chain reaction (PCR) allows rapid amplification of DNA fragments under the right conditions, resulting in many target DNA sequences, which is essential for biological research and clinical therapeutic aspects.
Each reaction component of PCR impacts the results, and annealing is critical to the high specificity of PCR. To ensure that you can successfully produce reproducible amplification products without non-specific amplification or contamination, you need to improve the chances of successful PCR experiments through proper primer design, reaction conditions, and a sterile environment.
Types of PCR
Conventional/Endpoint PCR
This is the most economical type of PCR reaction and can be performed directly using a thermal cycler followed by sequence detection using gel electrophoresis. When testing only for the presence of DNA sequences in a sample, conventional/endpoint PCR can give a quick answer in a few minutes.
Multiplex PCR
Multiplex PCR is characterized by using multiple primers, enabling researchers to obtain multiple targets simultaneously in the same PCR reaction. Multiplex PCR often requires extensive optimization of the annealing conditions to achieve maximum amplification efficiency with different primer-template systems. It is a cost-effective way to reduce the sample required, thereby increasing throughput and saving time compared to individual PCR experiments.
Rigorous hot-start procedures and specially optimized buffer systems are essential for the success of multiplex PCR. Therefore, to perform successful multiplex PCR experiments, the experimenter needs to plan the primer design and reaction conditions before the experiment to minimize primer cross-reactivity and non-specific binding. In addition, you need to coordinate the unwinding temperatures of different primers to ensure they can amplify the target sequences simultaneously.
Long-range PCR
Long-range PCR can amplify longer DNA sequences than conventional PCR, typically from 4 kb to tens of kilobases. Frequent optimization is required during amplification, especially for PCR products longer than 4 kb. The primers should be specific enough, and the length and Tm values should be sufficiently high. The primers are also desirable to be of sufficient specificity, length and Tm value to ensure stable binding to the target sequence.
Single-cell PCR
Single-cell PCR involves amplifying individual cells on PCR to obtain information on cell types and dynamic changes during development. Flow cytometry or microscopic manipulation can isolate individual cells of interest based on cell surface markers or physical appearance.
Fast-cycling PCR
As the name suggests, Fast-cycling PCR allows PCR reactions to be completed in a shorter period, shortening experimental preparation time and allowing researchers more time for downstream analysis. Fast-cycling PCR requires optimized reaction conditions and highly optimized reagents to ensure amplification specificity and sensitivity. It is susceptible to exogenous DNA contamination, so reacting in a sterile environment is essential.
Methylation-specific PCR (MSP)
MSP can determine the methylation status of the target DNA following bisulfite treatment. The method requires the design of two sets of primers: one set to anneal to unaltered cytosine (i.e., methylated in genomic DNA) and one set to anneal to uracil produced as a result of bisulfite treatment of unmethylated cytosine in genomic DNA.
You must use strict and highly specific PCR conditions to avoid non-specific primer binding and amplification of PCR artifacts. This is particularly important. Because converting non-methylated cytosine to uracil reduces DNA complexity and increases the likelihood of non-specific primer-template binding.
Hot start PCR
Hot start PCR application should block the polymerase activity before starting, which can effectively reduce the occurrence of non-specific amplification. It is suitable for detecting low-abundance target sequences, such as rarely present mutant genes or trace amounts of pathogen DNA.
High-fidelity PCR
High-fidelity PCR is suitable for amplifying longer DNA templates, and using high-fidelity polymerases is fundamental to effectively reducing the error rate in the reaction.
RAPD: Rapid amplified polymorphic DNA analysis
RAPD is a PCR-based tool for studying organisms at the molecular level. It uses small, non-specific primers to amplify seemingly random regions of genomic DNA. When analyzed on agarose gels, successful primer pairs produce different band profiles of PCR products between individuals, strains, species, etc. In RAPD, primers are used to amplify the DNA of the genome.
In RAPD, primers are only about 10 bases long. Therefore, annealing temperatures of <40°C are required.
RAPE: Rapid amplified polymorphic DNA analysis
RACE is a variant of RT-PCR. It is usually used to clone the remaining incomplete cDNA. There are two general techniques:
5' RACE - amplification of the end of the 5' cDNA
3' RACE - amplification of 3' cDNA ends
The first step, the same for both RACE types, involves converting the RNA to single-stranded cDNA using reverse transcriptase.
The second step is unique to each type of RACE, although each produces information that may yield a full-length cDNA sequence.
Because RACE uses an "anchor site" within the mRNA as a reference point, it is sometimes called "anchored PCR."
In Situ PCR
In situ, PCR can identify cellular markers and further localize cell-specific sequences within a cell population. It is a powerful tool in applications such as disease progression studies.
The procedure can use fresh or fixed cell or tissue samples, but sample preparation is critical to the results as fixation directly affects the PCR signal. The procedure suits radiolabelled, fluorescently labeled, or biotin-labeled nucleic acid probes.
Differential Display PCR
Differential display PCR compares and identifies differences in mRNA (and gene) expression patterns between two cell lines or populations.
The technique was invented in the 1990s and quickly became a key tool for gene expression analysis. However, it has recently been superseded by RNA-seq, microarrays and qRT-PCR.
Digital and Digital Droplet PCR (dPCR/ddPCR)
Although less common, this type of PCR reaction is often used in applications with complex or rare samples. dPCR divides the DNA sample into several compartments, each most likely to contain a single sequence. This analysis estimates the amount of starting material in the original DNA sample.
Quantitative PCR
Quantitative PCR (qPCR) is also known as real-time PCR. A real-time thermocycler records the fluorescence signal during each PCR cycle in qPCR.
Different between qPCR and dPCR
qPCR allows for the relative quantification of target DNA and provides a reliable, established method to test for the presence or absence of specific sequences.
dPCR uses a similar chemical method to detect DNA sequences and is performed in many tiny volumes or droplets, using the power of maths to improve the signal-to-noise ratio. Here is a brief comparison of the two:
| Feature | Digital PCR (dPCR) | Quantitative PCR (qPCR) |
|---|
| Principle | Partitioning of the sample into thousands of tiny partitions; end-point analysis | Continuous amplification of DNA with real-time fluorescence detection |
| Amplification | Each partition contains a single or few copies of DNA; amplification occurs within each partition | Continuous amplification until fluorescence signal reaches a detectable threshold |
| Sensitivity | Higher sensitivity due to absolute quantification and detection of rare events | Lower sensitivity due to potential for amplification biases and inhibition |
| Precision | Higher precision as absolute quantification is achieved for each partition | Moderate precision due to the variability in amplification cycles |
| Dynamic range | Broader dynamic range due to absolute quantification across a wide range of concentrations | Limited dynamic range due to exponential amplification |
| Quantification accuracy | Higher accuracy for absolute quantification and reduced susceptibility to PCR inhibitors | Slightly lower accuracy due to potential for and reduced susceptibility to PCR inhibitors |
| Quantification units | Copies/μL or copies per partition | Cycle threshold (Ct) values |
| Application | Ideal for rare mutation detection, absolute quantification, and digital counting | Widely used for gene expression analysis, SNP genotyping, and pathogen detection |
| Multiplexing | Limited multiplexing capabilities | Higher multiplexing capabilities with fluorescent probes |
| Cost | Generally higher cost due to specialized equipment and reagents | Generally lower cost due to widespread availability of equipment and reagents |
Enzymes used in PCR
There are several key enzymes that play an important role in the polymerase chain reaction (PCR). The following are the enzymes that are primarily used in the PCR process:
Taq DNA Polymerase
Taq DNA polymerase is the most classical and commonly used enzyme in PCR reactions. It can stabilize its activity at high temperatures and is suitable for many PCR assays. Setting up the reaction on ice is necessary to avoid non-specific amplification during the reaction.
DNA polymerases used in PCR
| Enzyme properties | DNA polymerase
family A | DNA polymerase
family B
|
|---|
| Available enzymes | Taq DNA polymerase | Proofreading enzymes
|
| 5'–3' exonuclease activity | + | – |
| 3'–5' exonuclease activity | – | + |
Extension rate
(nucleotides/second) | ~150 | ~25
|
| Error rate (per bp/per cycle) | 1 in 103 / 104 | 1 in 105 / 106
|
| PCR applications | Standard, hot-start, reverse transcription, real-time | High fidelity, cloning, site-directed mutagenesis |
| A-addition | + | Sometimes |
Hot Start DNA Polymerase
When the amplification reaction is set up at room temperature, primers can bind non-specifically to each other to form primer dimers. During the amplification cycle, the primer dimers can extend to produce non-specific products, which reduces the yield of specific products.
Hot-start PCR is critical for successful specific results for more challenging PCR applications. To generate hot-start DNA polymerase, antibodies can inhibit Taq DNA polymerase activity at lower temperatures.
In contrast, during antibody-mediated hot start, the antibody binds to the polymerase through relatively weak non-covalent forces, which leaves some polymerase molecules in an active state. This sometimes leads to further amplification of non-specific primer extension products during PCR. When run on agarose gels, these products show up as smeared or incorrectly sized fragments.
High Fidelity DNA Polymerase
High Fidelity PCR Enzymes typically provide 3' to 5' nucleic acid exonuclease activity to remove misincorporated bases. High-fidelity PCR Enzymes are well-suited for applications that require low error rates, such as cloning, sequencing, and targeted mutagenesis. However, if the enzyme is not provided in the hot-start version, the 3' to 5' Nucleic Acid Exonuclease Activity may degrade the primers during PCR setup and early stages of PCR. Non-specific triggering caused by shortened primers may result in trailing on the gel or amplification failure - especially if a small amount of template is used.
The proofreading function often causes the high-fidelity enzyme to work more slowly than other DNA polymerases. In addition, the A-addition function required for direct UA or TA cloning is greatly reduced, resulting in the need to ligate and transform less efficient flat-end clones.
Reverse Transcriptase
Used in reverse transcription polymerase chain reaction (RT-PCR) to transcribe RNA into the corresponding DNA. Commonly used reverse transcriptases include M-MLV Reverse Transcriptase and AMV Reverse Transcriptase.
Thermolabile DNA Polymerase
They are used in the final stage of the PCR reaction to remove possible residual DNA contamination. These enzymes are inactive during the routine PCR step but are active during the final amplification step.
PCR Primer Design
The optimal primer sequence and appropriate primer concentration are critical for maximum specificity and efficiency of PCR.
Primer Sequence:
Avoid 3-nucleotide parsimony at the 3' end, i.e., use Met or Trp coding triplets at the 3'.
To improve primer-template binding efficiency, reduce concatenation by allowing some mismatches between primer and template, especially at the 5' end, but not at the 3' end.
Design primers with less than 4-fold parsimony at any given position.
Primer concentration:
Begin PCR with a primer concentration of 0.2 µM.
If PCR efficiency is poor, increase the primer concentration in 0.25 µM increments until satisfactory results are obtained.
Guidelines for the design and use of primers
| Standard PCR | Multiplex PCR | One-step RT-PCR
|
|---|
| Length | 18–30 nt | 21–30 nt | 18–30 nt
|
| GC content | 40–60% | 40–60% | 40–60%
|
| Tm information | The Tm of all primer pairs should be similar | The Tm of all primer pairs should be similar. For optimal results, the Tm should be 60–88°C | The Tm of all primer
pairs should be similar.
The Tm should not be
lower than the temperature of the reverse transcription (e.g., 50°C)
|
| Estimating optimal annealing temperature | 5°C below the calculated Tm | 5–8°C below the calculated Tm (when greater than 68°C) or 3–6°C below the calculated Tm (when 60–67°C) | 5°C below the calculated Tm |
| Location | – | – | To prevent detection of gDNA:
Primer hybridizes to the 3' end of one exon and the 5' end of the adjacent exon. Alternatively, the primer hybridizes to a flanking region that contains at least one intron.
If only the mRNA sequence is known, choose primer annealing sites that are
300–400 bp apart. |
| Concentration, A260 unit equivalence | 20–30 µg | 20–30 µg | 20–30 µg |
PCR Troubleshooting Guide
PCR experiments. If you encounter practical results that do not always meet expectations, you know what the reason? Next, we will discuss the problems that may occur in the use of PCR and troubleshooting techniques:
| Causes | Solutions |
|---|
DNA Template
| Poor integrity | 1. When isolating DNA, minimize cutting or incising of the DNA. 2. The integrity of template DNA can be checked by gel electrophoresis. 3. Store DNA in molecular grade water or TE buffer (pH 8.0) to prevent degradation by nuclease.
|
Low purity
| 1. If a chemical or enzymatic DNA purification protocol is used, ensure no PCR inhibitors remain. 2. Repurify or precipitate and wash DNA using 70% ethanol to remove residual salts or ions that may inhibit DNA polymerase.
|
| Concentration imbalance | Prepare fresh deoxynucleotide mixture |
Underuse
| 1. Increase the starting volume appropriately. 2. Select DNA polymerase with high sensitivity for amplification. 3. Increase the number of PCR cycles appropriately.
|
| High GC content or secondary structure | 1. Select DNA polymerase with high synthetic capacity. 2. Use PCR additives or co-solvents to promote the denaturation of GC-rich DNA and sequences with secondary structures. 3. Increase denaturation time or temperature to dissociate double-stranded DNA templates efficiently.
|
| Long fragment | 1. Confirm the long fragment amplification capability of the selected DNA polymerase. 2. Use a DNA polymerase designed for long fragment PCR. 3. Select a DNA polymerase with high synthetic capacity. 4. Lower the annealing and extension temperatures to promote primer binding and improve enzyme thermal stability. 5. Increase the extension time according to the amplicon length.
|
| Template DNA is damaged | Repair the DNA template to limit UV exposure when analysing or excising PCR products from the gel. |
| Required sequence may be toxic to the host | Cloning into non-expression vectors uses low copy number cloning vectors. |
| Primer | Problematic design
| 1. Modify the primer design. 2. Use an online primer design tool. 3. Ensure that the primers are specific for the target fragment. 4. Confirm that the primer complements the correct target DNA strand.
|
| Primer obsolescence | 1. Resuspend and dispense primers and store them properly. 2. Dispense fresh primers or purchase new primers as needed.
|
| Insufficient dosage | 1. Optimise the primer concentration (usually in the range of 0.1-1 μM). 2. For long fragment PCRs and PCRs with concatenated primers, the minimum starting concentration is 0.5 μM.
|
| Inappropriate DNA polymerase | 1. Using hot-start DNA polymerase prevents primer degradation due to the corrected DNA polymerase's 3'→5' nucleic acid exonuclease activity. 2. Prepare the PCR reaction system on ice or add the DNA polymerase to the reaction mixture.
|
| Insufficient amount of DNA polymerase | 1. Select a DNA polymerase with high sensitivity for amplification.
2. Check the recommended amount of DNA polymerase for PCR and optimize as necessary.
3. Increase the amount of DNA polymerase used. |
| Insufficient Mg2+ concentration | Optimise the MG2+ concentration to obtain the highest PCR yield. |
| Excessive PCR additives or co-solvents | 1. Use the recommended concentration of co-solvent. 2. Use the lowest concentration possible. 3. Adjust the annealing temperature. 4. Increase the amount of DNA polymerase or use a DNA polymerase with high synthesis capability. 5. Consider using special additives or co-solvents for the specific DNA polymerase.
|
| Uneven reagents | Mix well to eliminate density gradients that may develop during preservation and reaction preparation. |
| Thermal cycling conditions | Unsatisfactory denaturation results | 1. Optimise DNA denaturation time and temperature.
2. Short denaturation times and low temperatures may not result in good double-stranded DNA template dissociation. Conversely, long denaturation times and high temperatures may decrease enzyme activity. |
Unsatisfactory annealing results
| 1. Use a gradient cycler whenever possible to progressively optimize the annealing temperature in 1-2°C increments.
2. The optimal annealing temperature is usually 3-5°C lower than the lowest primer Tm.
3. Annealing temperature should be adjusted when using PCR additives or co-solvents. Annealing temperatures for primer pairs vary depending on the DNA polymerase. |
| Unsatisfactory extension | 1. Select an extension time appropriate for the length of amplification.
2. lowering the extension temperature (e.g., to 68°C) maintains enzyme activity when amplifying long fragments (e.g., >10 kb).
3. Use a DNA polymerase with high synthesis capacity to achieve stable amplification even at short extension times. |
| Inappropriate number of PCR cycles | 1. adjust the number of cycles (usually 23-35 cycles)
2. Increase the number of cycles to 40 if the DNA starting amount is less than 10 copies. |
| Insufficient denaturation | The denaturation time or temperature may be increased to allow efficient dissociation of the DNA. |
| Wrong annealing temp | Select the appropriate annealing temperature according to the different DNA polymerases. |
| Annealing temp too low | 1. Increase annealing temperature to improve specificity.
2. The optimal annealing temperature is usually 3-5°C lower than the lowest primer Tm.
3. Use a gradient cycler to optimize the annealing temperature in 1-2°C increments.
4. Consider using landing PCR to improve specificity. |
| Too long annealing time | Shorten the annealing time to minimise primer binding to non-specific sequences. |
| Extension temperature too high | Reducing the extension temperature by 3-4°C improves the thermal stability of DNA polymerase, especially for long fragment PCR. |
| Insufficient extension time | 1. When amplifying longer DNA fragments, increase the extension time. |
| Too many cycles | 1. Reduce the number of cycles without significantly reducing the yield of the target PCR product and prevent the accumulation of non-specific amplicons. |
| Sequence errors in PCR products | Problems with primer design | Avoid direct repeats in primer sequences |
| Low product quality | 1. Purchase PCR primers that have been purified to remove non-full-length DNA oligonucleotides that are truncated at the 5′ end. |
| Nuclease contamination | 1. use molecular grade, nuclease-free reagents to formulate the PCR reaction system.
2. Formulate the reaction system on ice to keep possible contaminating nucleic acid exonucleases as low as possible. |
| UV damage to DNA | 1. Use a long-wave UV (360 nm) lightbox to observe the fragments in the gel, keeping the exposure time as short as possible.
2. If using a short-wave (254-312 nm) light box, reduce the UV exposure time to a few seconds and place the gel on a glass or plastic plate. |
| Sequencing errors | 1. Sequence the DNA double strand to verify the reliability of the sequencing results.
2. Samples are taken in duplicate as far as possible. |
| False-positive amplification | Cross-contamination | 1. Use pipette tips with an aerosol barrier.
2. Designate a separate work area and decontaminate the area after each use.
3. Use techniques to prevent residual PCR contamination, such as dUTP doping and UDG treatment. |
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