An Overview of PCR Methods in Clinical Diagnosis and Biology Research

History

Polymerase Chain Reaction (PCR) is a widely used technique, extensively applied in clinical diagnosis and molecular biology research. PCR is the amplification of specific nucleic acid (NA) sequences by DNA polymerase in vitro. The leap in PCR technology occurred in 1983 when Kary Mullis popularized the use of thermostable polymerase accompanying temperature cycling. The universal application of PCR lies in its ability to amplify a small quantity of target nucleic acid sequences into a large amount of PCR products, which can then be detected by downstream methods, such as observing nucleic acid (NA) products through agarose gel electrophoresis. This is due to the exponential amplification of nucleic acid (NA) sequences, resulting in the original template (target nucleic acid sequence) being amplified to produce millions of copies.

Basic PCR Reaction

PCR amplifies target nucleic acid sequences using DNA polymerase, primers, and nucleotides. The template for a PCR reaction can be any target nucleic acid sequence. The source of the nucleic acid can be DNA, RNA, or cDNA. Primers are short sequences of nucleic acids synthesized in vitro. Primers are designed to complementarily bind to the antisense strand of a specific target nucleic acid template, with the length of the primers usually between 15-40 bases. Primers should ideally lack secondary structure and not be complementary to each other to prevent the formation of primer dimers. Various DNA polymerases can be used in PCR, but the most widely used is the heat-resistant Taq DNA polymerase. This enzyme extends the primer according to the nucleic acid sequence of the template (adding dNTPs or nucleotides to the end of the primer).

PCR reactions typically consist of 20-40 temperature cycles. The denaturation of the nucleic acid template sequence is carried out at 95℃. Primer annealing and binding to the target sequence occur when the temperature is cooled to 37-60℃. The extension of the primer nucleotides is carried out by the DNA polymerase within the range of 60-72℃. Standard cycling conditions start with 95℃ for 5 minutes to denature all nucleic acid templates, followed by 2-40 repetitions of temperature cycles (95℃ for 30 seconds, 60℃ for 30 seconds, 72℃ for 1 minute). The duration at each temperature can be optimized for specific experiments. For example, amplification of very short target sequences requires much shorter durations at each temperature compared to very long target sequences. The target sequence product obtained in each temperature cycle is twice that of the previous cycle. This leads to exponential amplification of the original template, typically resulting in millions or billions of copies of the original target nucleic acid sequence.

Basic PCR reactions have three phases (Figure 1). The exponential phase is where the nucleic acid product of each temperature cycle is exactly doubled. Real-time PCR detection is carried out during the exponential phase. The linear phase occurs when the reaction slows down due to the consumption of reagents and the degradation of the product. The final phase is the plateau phase, where the reaction stops and no more amplification products are generated. Conventional PCR reactions are analyzed by gel electrophoresis at the plateau phase.

Figure 1. Phases of a PCR Reaction: During the PCR process, product doubling occurs in the exponential phase, product amplification slightly less than doubling occurs in the linear phase, and very low, almost stagnant product amplification occurs in the plateau phase.

Downstream Detection of PCR Reactions

There are many ways to perform downstream detection of PCR products. A commonly used observation method is through agarose gel electrophoresis. This involves separating nucleic acid fragments by electrophoresis, staining nucleic acid fragments with embedded dyes such as ethidium bromide or SybrSafe, and then detecting with a UV light source and imaging system (Figure 2).

Figure 2. Photo of DNA Agarose Gel: The far-left row is a DNA ladder, containing nucleic acid fragments of known sizes. The bottom band on the gel is a nucleic acid fragment 100 base pairs in length. The rows to the right are PCR amplification products 120 base pairs in length.

Real-time PCR uses a specialized thermal cycler to detect the fluorescence signal in each reaction well. This signal indicates the amount of double-stranded nucleic acid contained in the reaction tube or well. This signal is expressed as relative fluorescence units and is plotted by the thermal cycler software relative to the number of temperature cycling cycles (Figure 3).

Figure 3. Real-Time PCR Amplification Curve: The graph correlates the fluorescence signal with the number of temperature cycles. This signal is measured in real time, allowing for the monitoring of the reaction's progress as the PCR proceeds.

Applications

PCR is widely used in scientific research, clinical, and forensic fields. In molecular biology research, PCR is commonly used for genetic engineering, DNA sequencing, and gene expression analysis. In clinical laboratories, PCR is crucial for the detection of infectious diseases. In forensic identification, PCR applications include paternity testing and DNA fingerprinting. These applications all take advantage of the high sensitivity of PCR, which can detect target nucleic acid sequences even from a single human hair. Table 1 lists the main applications of PCR.

Table 1: Major PCR Applications

Methods

Standard/Conventional PCR

There are many types of PCR methods, each with its own advantages and limitations. Standard or conventional PCR is the most basic type of PCR reaction. It provides qualitative results and requires DNA detection or observation steps after PCR is completed. The main advantage of conventional PCR is its relatively low cost, and almost all laboratories have available conventional PCR equipment. Typically, after a conventional PCR reaction is completed, products are separated by agarose gel electrophoresis according to the size of nucleic acid fragments. DNA amplification products are observed using embedded dyes such as ethidium bromide or Sybr Safe and a UV light source. Preliminary determination of PCR reaction specificity is through the size of product fragments compared to a DNA ladder, a DNA ladder is a mixture of DNA fragments of known sizes. The product's electrophoresis bands can be cut and separated from the gel, and the DNA in the bands is purified and sequenced to more reliably determine the PCR reaction's specificity. Figure 2 is an example of an agarose gel electrophoresis diagram, with a DNA ladder on the left and electrophoresis of several PCR reaction products on the right. The limitation of standard or conventional PCR is its low sensitivity, and it cannot provide quantitative results.

Table 2: Main PCR Methods, and Their Pros and Cons

Real-Time PCR

Real-time PCR is a more recent, but now very common, PCR method that has several advantages over conventional PCR. First, it allows for real-time detection of PCR products, eliminating the need for post-PCR agarose gel electrophoresis for DNA visualization. In addition, real-time PCR is quantitative and specific. The initial quantity of the target sequence in the sample can be determined by comparison with a standard curve of known quantities of DNA. The specificity of the PCR reaction is enhanced by the use of specific nucleic acid probes and/or melt curve analysis after the PCR reaction. Refer to Figure 4 for an example of a melt curve for a single PCR product. The presence of a single peak indicates a single amplification product from the PCR reaction. The presence of multiple peaks indicates multiple amplification products from the PCR reaction, suggesting that the PCR detection system needs further optimization and improvement. The limitations of real-time PCR are the increased cost and the need for specialized thermocyclers, as well as the limited precision in quantifying the initial quantity of target nucleic acid sequences.

Figure 4. Melting Curve Graph: After real-time PCR detection using DNA binding dyes, the reaction products are analyzed by melt curve analysis to determine if a single PCR product has been generated.

PCR Array

PCR arrays are used with real-time PCR thermocyclers and are based on SYBR green detection in real-time PCR. PCR arrays involve adding different gene-specific primers to each well of a 96-well plate, allowing for the expression of up to 88 genes in the same sample to be detected, as well as eight standard or control reactions. Each well of the 96-well plate in a PCR array detects a specific product of a single gene. PCR arrays typically include control reactions that provide quantitative results for gene expression. PCR arrays are typically used for expression analysis of a set of related genes in a specific biological pathway (e.g., DNA repair, cell cycle, or oxidative stress). The advantage of PCR arrays is that they can simultaneously detect the expression of multiple different genes in one sample. The limitation is that each sample must be processed separately, which is very time-consuming and also expensive.

Microfluidic Chip PCR

Microfluidic chip PCR uses microfabrication and microfluidics and can amplify DNA more quickly than conventional or real-time PCR. In addition, microfluidic chip PCR is small in size and integrates detection elements, making it more portable and suitable for field use. Digital and droplet digital PCR can separate nucleic acid molecules and quantify PCR end products without the need for a standard curve. This allows for very precise measurement of copy numbers and the detection of very low copy numbers of target nucleic acid sequences. While microfluidic chip PCR is powerful and precise, it is unaffordable for many researchers. This is due to the necessary microfabrication equipment or the need to purchase prefabricated chips, which are quite expensive. However, microfluidic chip PCR is rapidly developing, and costs may soon decrease significantly.

Other PCR Methods

Many other PCR methods are also used in biological research. Genotyping typically uses allele-specific PCR. Epigenetic research primarily uses methylation-specific PCR. This technique detects methylation of CpG islands in genomic DNA. Touchdown PCR reduces non-specific amplification by gradually reducing the annealing temperature as the reaction progresses. This technique is beneficial for increasing the yield of specific target sequence amplification.

Challenges in PCR Methodology

Equipment

Although PCR is a widely used technique, there are still many opportunities for improvement to shorten the reaction time and reduce the volume of reaction fluids. The reaction speed depends on the type of heating and temperature cycling mechanism, as well as the volume of the reaction fluid, thus it is the primary target for PCR improvements. PCR systems based on metal block heating, which use indirect conduction heating, are common, but the thermal mass of the metal block is high, and the required reaction fluid volume is relatively large, so the rate of temperature change is relatively slow (3ºC/s). Roche Lightcycler uses convective heating and requires only a few microliters of reaction fluid volume, so the speed of temperature change is very fast (10ºC/s). Microchip PCR devices further accelerate the reaction speed and reduce the volume of reaction fluids. These chips are most commonly made of silicon or glass. These chips are thermoelectrically heated, heated by a rotating platform based on convective heating, or by embedded resistance heaters, and require only a few microliters of reaction fluid volume. Microchip PCR can also use direct infrared radiation from tungsten filament lamps for heating. It can also use direct infrared guidance from lasers for heating. Both methods can yield PCR results within minutes and require only a few picoliters of reaction fluid volume.

Experimental Errors

False-positive and false-negative PCR results affect the validity of PCR detection, so it is necessary to optimize PCR reactions to prevent these artefacts. False-positive results refer to the detection of DNA amplification products even when no starting template was added to the reaction system. This may be due to contamination. It is necessary to operate in a clean laboratory to prevent inadvertent contamination of the reaction system by aerosolized DNA. False-negative results refer to the inability to detect amplification products even though the target nucleic acid is present. This may be due to problems with the primers, or non-optimized temperature cycling parameters, or the amplified product quantity being below the detection limit of the system. In order to reduce the occurrence of false negatives, it is necessary to optimize the design of the PCR detection system and temperature cycling conditions. This includes selecting high-quality primers, optimizing temperatures during cycling, and using high-quality nucleic acid templates.

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Real-Time PCR

Advantages

Real-time PCR has significant advantages over conventional PCR. Real-time PCR uses a fluorescent indicator measured by the detection system, typically DNA amplification products in closed tubes of a 96-well plate. Real-time PCR does not require agarose gel electrophoresis for detection after PCR, significantly shortening the time required to obtain results. PCR products are detected after each temperature cycle, which allows for the measurement of PCR reaction kinetics. Compared to DNA detection through agarose gel electrophoresis, the fluorescence detection of PCR products is more sensitive, so a two-fold difference in target DNA can be detected by real-time PCR, but not by conventional PCR and agarose gel. In addition, using probe-based real-time detection, PCR amplification product detection is sequence-specific.

Detection of PCR Products

DNA Binding Dyes: Real-time PCR uses fluorescent dyes or probes that interact with PCR products. The two main types of fluorescent detection are DNA binding dyes, such as SYBR Green, or fluorescently labeled sequence-specific probes, such as TaqMan or Molecular Beacon probes. When DNA binding dyes bind to any double-stranded DNA fragment, the dye emits a fluorescent signal. When only single-stranded nucleic acids are present, these dyes do not bind to nucleic acids, and the fluorescent signal emitted is very weak. While SYBR Green is the most commonly used, there are also other DNA binding dyes in use. These dyes include SYTO 9, SYTO-13, SYTO-82, and EvaGreen. Because the detection of these dyes' fluorescent signals is not sequence-specific, melt temperature analysis must be performed to ensure that the PCR product is a single product. If the melt curve has multiple peaks, it indicates that multiple PCR products are present and the PCR detection system needs further optimization. Although this method can be time-consuming, SYBR Green and other DNA binding dyes are still popular because dye-based detection methods are significantly cheaper than probe-based detection methods.

Nuclease-Dependent Probes: The second major type of detection chemistry used in real-time PCR are sequence-specific fluorescently labeled probes. These probe systems can be nuclease-dependent probes or simple hybridization probes. Nuclease cleaved probes include TaqMan, HybProbe (two oligonucleotides), Minor Groove Binding (MGB) probes, and Locked Nucleic Acid (LNA) probes. These probes bind complementarily to the target nucleic acid sequence in the PCR amplification product, and a reporting fluorescent dye and a quenching fluorescent dye are covalently connected at both ends of the probe. These systems use Fluorescence Resonance Energy Transfer (FRET) technology. When the dyes are close to each other, the reporting dye's fluorescence signal is quenched, so no signal is detected. However, when the dyes are separated, the reporting dye's fluorescence is not quenched, so a signal can be detected. The sequence to which the probe anneals is within the PCR primer binding site, and as Taq DNA polymerase extends the primer to produce PCR products, its 5' exonuclease activity cleaves off the end of the probe. Once the quenching dye is removed, the reporting dye can produce a fluorescent signal upon excitation. Cycle Probe Technology (CPT) probes contain RNA nucleotides, so they are different from TaqMan type probes. After the CPT probe hybridizes to the target sequence, it forms an RNA-DNA double strand. Then the RNaseH enzyme cuts off the probe's quenching dye.

Hybridization Probes: Although hybridization probes are also sequence-specific, they do not require a nuclease to cut off the dye from the hybridization probe. Hybridization probes include Molecular Beacons, which have a single loop region between two reverse repeated sequences, forming a hairpin structure. The probe denatures and then anneals to the target sequence, opening the hairpin, separating the fluorescent dyes, and allowing the reporting dye to generate an enhanced fluorescence signal. Other detection probe technologies include Scorpions (probe and primer combined in one molecule) and LUX (Light Upon eXtension) detection. The hairpin structure of the LUX primer quenches the fluorescent dye signal at the 3' end of the LUX primer probe. Once the primer binds to the target sequence, as the DNA polymerase extends the primer sequence, the dye's fluorescence signal is enhanced.

Analysis of the results of real-time PCR

The fluorescence intensity detected by the real-time PCR thermal cycler system is proportional to the accumulation of PCR amplification products. However, the analysis of results is limited to data from the index period of the real-time PCR reaction, as this is the best data point for accurate quantification. The threshold cycle number (CT) is the number of cycles of temperature cycling when the fluorescence intensity in the real-time PCR reaction tube exceeds the threshold, which is set above the baseline but within the exponential period of amplification. Real-time PCR generally uses two quantitative assays. Relative quantification involves comparing samples from the experimental treatment to those from the control and determining the relative proportion of gene expression of the target sequence compared to the normalized expression of the internal reference gene. The key point is that the test treatment method does not have an impact on the expression of the internal reference genes used. The main advantage of relative quantification is because there is no need to establish a calibration curve, so assay time is reduced. Commonly used methods include the relative standard curve method, and the comparative CT method (ΔΔCT). See Pfaffl for the mathematical model calculation of the latter.

Absolute quantification detects the actual starting amount of target sequence in a sample by comparing the amplification signal of the sample to a standard curve of target DNA. The calibration curve can be based on a dilution of a nucleic acid molecule (either recombinant plasmid DNA in which a subclonal amplification sequence is inserted, a real-time PCR product, or a synthetic large fragment of oligonucleotide). Plasmid DNA is widely used because it is more stable than PCR products or oligonucleotides. However, shorter templates also require less time to prepare. The standard curve of the plasmid was obtained by performing real-time PCR on a known concentration of plasmid (inserted with subclonally amplified sequences), and the plot was drawn to correlate the logarithm of the CT value with the starting copy number of the target sequence (Figure 5). The CT value is inversely proportional to the logarithm of the starting copy number. The starting copy number of the target sequence of the test sample was determined by linear regression on the standard curve.

Figure 5. Standard curve for real-time PCR of plasmid DNA: Amplicon sequence of the recombinant cloned target gene of plasmid pGEMT. The presence of the target gene amplification sequence was confirmed by DNA sequencing. Dilute the plasmid in triplicate and draw a graph correlating the threshold cycle (CT) of the PCR reaction with the known starting amount of plasmid DNA.

Microfluidic Chip

Since 1990, innovations in miniaturization of PCR reactions and thermal cyclers are reflected in microfluidic chip PCR. for example, in 1998, Northrup et. al reported their creation of a micro analytical thermal cycler (MATCI), which is made of silicon and has integrated heaters in the reaction wells. The instrument is equivalent to a briefcase in terms of portability. Since then, the field of microfluidic PCR has taken off, with technological advances including a variety of processing materials and configurations.

The reaction chamber of most PCR chips is made of silicon. Silicon has excellent thermal conductivity and allows for rapid temperature changes during temperature cycling. However, chip materials can also be glass, polymers including polycarbonate and polydimethylsiloxane, ceramics, and 317 stainless steel. Chip construction can be based on static fixed reaction cavities or dynamic continuous flow. With fixed reaction chamber chips, the PCR reaction solution is static and the reaction chamber undergoes temperature cycling. These chips may be single-chamber or multi-chamber. Dynamic continuous flow chips, where the sample is pump driven through the microchannels during the temperature cycling of the PCR amplification. The sample flow rate of these chips determines the time of temperature transition. Microchip PCR reduces assay time, low reagent consumption, fast temperature cycling for heating and cooling, and reduced power consumption. Microchip PCR may require downstream processing to detect DNA, such as agarose gel electrophoresis or capillary electrophoresis. However, on-chip DNA detection methods such as capillary electrophoresis, fluorescence detection or DNA hybridization methods may also be included. Microchip PCR technology continues to evolve, including the integration of microchip detection systems, and the optimization of processing materials and temperature cycling methods.

Digital and Droplet-based Digital PCR

The microfluidic chamber-based digital PCR (cdPCR) method enables absolute quantification of nucleic acids without the use of a standard curve. Digital cdPCR is where the sample is separated into discrete droplets of water-in-oil, which may not contain any target nucleic acid or may have one or more copies of the target nucleic acid sequence. After PCR amplification, the presence of the target sequence is detected and the concentration is calculated by counting the percentage of positive droplets out of the total number of droplets. "Droplet" digital PCR is the most sophisticated digital PCR method. "Droplet" digital PCR has 25 times more droplet separations than digital PCR, allowing for very accurate estimation of the copy number of target sequences.

References

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