Apr. 29, 2024
While the basic PCR procedure is still commonly used in many laboratories, PCR has evolved far beyond simple amplification and detection. Many variations of the original PCR method have been described, which include:
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4.1 Hot Start PCR
In conventional PCR, nonspecific primer annealing can occur at low temperatures, and although the activity of the DNA polymerase is significantly compromised, extension can still occur. This results in undesired PCR products and lower yields of desired product. Hot start PCR involves blocking DNA polymerase activity at low temperatures and is used to reduce nonspecific amplification and improve the performance of PCR. Hot start PCR can be achieved by simply withholding the polymerase until higher temperatures are attained. Other methods include using physical barriers, such as wax beads, to segregate key reaction components, using specific inhibitors or antibodies to block DNA polymerase activity, and chemically modifying other components such as dNTPs and primers.
4.2 Reverse Transcriptase PCR
The DNA polymerases used for basic PCR require a DNA template. Numerous applications such as gene expression profiling, however, would benefit from amplification of RNA. The reverse transcriptase PCR (RT-PCR) was, thus, developed. In RT-PCR, the RNA sample must first be reverse transcribed to cDNA using a reverse transcriptase enzyme. The resulting cDNA can then be used for subsequent PCR amplification.
4.3 Quantitative PCR (qPCR)
Quantitative PCR (qPCR) enables accurate quantification of starting amounts of DNA, cDNA, and RNA targets. Unlike the conventional PCR where quantification occurs as a separate step after the amplification of DNA, in qPCR, a fluorescent reporter dye enables a real-time detection of PCR products during each amplification cycle. An increase in nucleic acid amplification is proportional to the increase in fluorescence signal. Because qPCR detects the amount of nucleic acids as the reaction progresses, it provides a wide, linear, dynamic range; demonstrates high sensitivity; and is very quantitative as its name suggests. Frequently, qPCR is combined with reverse transcriptase PCR to quantify messenger RNA, microRNA and non-coding RNA.
4.3.1 qPCR Fluorophores
Reporters in qPCR are either (1) DNA-binding dyes or (2) fluorescently-labeled probes.
Double-stranded DNA (dsDNA)-binding dyes bind reversibly, but tightly to dsDNA by intercalation and/or minor groove binding. Before amplification, during the presence of single-stranded DNA, these dyes emit very low intensity signals that are undetectable. However, at the end of the elongation step, as dsDNA levels increase, the dyes bind to dsDNA and fluoresce high-intensity signals.
Among the commercially available fluorescent dyes, the SYBR® Green is by far the most widely used dsDNA-specific dye for real-time PCR. SYBR Green binds all dsDNA molecules, emitting a fluorescent signal of a defined wavelength on binding.
DNA binding dyes cannot be used for multiplex reactions because fluorescent signals from different amplicons cannot be distinguished from one another. Parallel reactions can, however, be set up to examine multiple genes in a qPCR assay with SYBR Green.
Using dsDNA-binding dyes will provide the simplest and cheapest option for real-time PCR, but the principal drawback is that both specific and non-specific products generate signal. High PCR specificity is required when using fluorescent dyes, and a post-PCR dissociation (melting) curve analysis should be carried out to confirm that the fluorescence signal is generated only from target templates and not from the formation of non-specific PCR products.
This useful application note by Analytik Jena offers a qPCR performance comparison of six different ready-to-use master mixes with an intercalating dsDNA-binding dye for the qPCR of human GAPDH. It is interesting to note the differences in performance in the very same experiments by merely changing the reagents.
Figure.1 The real-time plots of qPCR reaction on human GADH using six different master mixes with intercalating dyes. Download the method here.2. Fluorescently-labeled probes
Fluorescently-labeled probes bind to a specific region of the template DNA and provide a highly sensitive and specific method of detection. Several probes based on different chemistries are available for real-time detection; these include:
I. TaqMan® probes:
TaqMan hydrolysis probes employ the 5’ → 3’ exonuclease activity of Taq. Carrying a fluorophore and a quencher, they bind to a specific oligonucleotide sequence of the template DNA. As the probe binds to a pre-determined region of the DNA template; the fluorophore is attached at the 5' end of the probe and the quencher dye is located at the 3' end. When intact during annealing, the fluorophore’s signal is quenched by the close proximity of the quencher. During amplification, the dsDNA-specific 5' → 3' exonuclease activity of Taq cleaves off the reporter. Now separated from the quencher, the reporter fluoresces, its signal proportional to the amplified product.
HOW-TO-BUY TIP: Select quenchers that are compatible with the fluorophore selected. For optimal performance, the quencher’s absorbance spectrum should match the fluorophore’s emission spectrum as closely as possible.
II. Molecular beacons:
Molecular beacons are dual-labeled probes with a fluorophore attached at the 5' end and a quencher dye attached at the 3' end. The probes are designed such that the ends have complementary sequencing. Therefore, when in solution, the two ends of the probe hybridize and form a hairpin structure. With the fluorophore and quencher in close enough proximity, the fluorescent signal is quenched. When the probe binds to the target sequence, the stem opens and the fluorophore and quencher separate. This generates a fluorescent signal in the annealing step that is proportional to the amount of PCR product. Molecular beacons add another level of specificity to real-time PCRs and are particularly useful for allelic discrimination experiments.
III. Dual hybridization probes:
These involve using two labeled oligonucleotide probes that are designed to bind to two adjacent regions on target DNA. The first probe carries a donor dye at its 3' end, while the second carries an acceptor dye at its 5' end. The donor and the acceptor dyes are selected such that they exhibit FRET (fluorescence resonance emission transfer). During annealing, excitation is performed at a wavelength specific to the donor dye, and the reaction is monitored at the emission wavelength of the acceptor dye. Fluorescence detected during the annealing phase of PCR is proportional to the amount of amplification produced.
HOW-TO-BUY TIP: It is important to select fluorescent labels that are compatible with the detection channels and filters of the real-time PCR instrument you are using.
4.3.2 Real-time thermal cyclers
A qPCR experiment requires a specialized real-time PCR thermal cycler equipped with fluorescence detection modules used to monitor the fluorescence as amplification occurs. Listed below are considerations for purchasing a real-time PCR thermal cycler:
The key feature of real-time PCR is precise fluorescence measurements, making optics a very important consideration when purchasing a qPCR thermal cycler. It is important that your thermocycler transmits homogeneous excitation and illumination across all your samples in, say, a 96-well experiment, for reproducibility and consistency. The qTOWER3 by Analytik Jena, for example, includes a patented fiber-optic shuttle system with a unique light source comprising of four different-colored (RGBW) LEDs, thus enabling excitation of all known fluorescent dyes in your experimental plan.
To save time and to ensure constant experimental conditions, it has become crucial to consider multiplexing PCRs. Find out how many different fluorescent-labeled probes can be used in your qPCR thermocycler. For instance, qTOWER3 offers multiplexing capabilities with six different probes ranging from blue to near-infrared wavelengths.
In addition to fluorescence detection in a qPCR experiment, the heat-enabled amplification itself determines the success of the experiment. Enquire about your desired instrument’s temperature control, i.e. if set at 65⁰C, how precisely is 65⁰C maintained across all wells? Plus, the ramping rate of the instrument – the time it takes for the instrument to change temperature settings – becomes a factor in completing the protocol faster. Analytik Jena’s qTOWER3, for example, exhibits a temperature control precision of ± 0.1⁰C and a ramping rate of 8⁰C/s.
With extreme temperature changes across an experiment, condensation can become an issue, often resulting in a loss of samples. Check if the qPCR device of your choice is equipped with a motorized, heated lid that curbs condensation.
Figure 2. Learn more about the features of qTOWER3 real-time thermal cycler from Analytik Jena in this video
Your experiment is only as good as the light detected from the fluorophores. Enquire about the detectors in the device of your choice, the read-out times and the compatibility of different color modules, i.e. the use of DNA-binding dyes, molecular beacons or dual hybridization probes with your experiments. A high-sensitivity photomultiplier tube in qTOWER3 detects signals with a read-out time of six seconds for 96 wells, irrespective of the number of dyes. With 12 color-, FRET- and protein-based modules in the qTOWER3, you can plan experiments with a diverse set of reporters. Plus, the detection module can accept up to six different color filter modules, enabling retrofitting for future developments.
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If your instrument is compatible with a 384-well temperature block, it instantly adds high-throughput capabilities to your experiments, miniaturizing individual reactions. The qTOWER3 84 is adapted to the 384-well format, with up to six-fold multiplexing capabilities, and contains 16 scanning fibers, enabling extremely fast read-outs in six seconds.
Many modern real-time thermal cyclers now possess an inbuilt software analysis system, coupled with a touch screen or computer integration. To instantly obtain results, consider a software package that calculates Ct values and provides graphs you can directly use. The easy-to-use qPCRsoft package in the qTOWER3 covers an entire spectrum of analyses and doesn’t require any additional licenses to use.
The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describes the minimum information necessary for evaluating real-time PCR experiments and helps promote better experimental practice. A practical guide to publishing data that conform to the MIQE guidelines can be read here. Enquire with the manufacturer if you can obtain a copy of the MIQE-compliant documentation of the instrument you are purchasing.
4.3.3 Applications of qPCR
Real-time PCR applications include gene expression analysis, validation of DNA microarrays, single nucleotide polymorphism (SNP) genotyping, copy number variation analysis, allelic discrimination analysis, drug metabolizing enzyme (DME) analysis, clinical molecular diagnostics, viral load quantification, and pathogen detection.
When analyzing gene expression using qPCR, some genes are expressed at very high copy numbers, while some others may be expressed at very low copy numbers. With lower copy numbers, obtaining equal-volume aliquots containing a copy of the gene becomes a challenge, whereas with very high copy numbers, the reagents don’t suffice or there’s too much background interference. This technical note by Analytik Jena details an experiment on the human GAPDH gene and demonstrates a wide linear range with accurate quantification across 10 orders of magnitude.
In the food industry, significant efforts are being made to detect adulteration or non-declared constitutes of animal origin in food in order to comply with regulatory standards and, in some countries, religious and health laws. Real-time PCR forms a convenient method to identify traces of animal DNA in food, for example, to determine the origin of gelatin in gummy bears or to identify traces of pork in rice. This useful downloadable application note outlines the types of qPCR experiments performed to test for sources of food products from different animals such as sheep, goat, cow and so on. The method also goes on to describe detection of food-borne pathogens, such as salmonella, using qPCR.
Figure3. (Left) Amplification plot for cow DNA in various milk sources and (right) amplification plot of salmonella DNA. Both experiments were performed and measured on the qTOWER³. Get complete access to the downloadable method here.4.4 Digital PCR
Digital PCR (dPCR) is a technology that provides absolute counts of target DNA, as well as increased sensitivity, precision and reproducibility, compared to qPCR. dPCR has the potential to have a major impact on molecular analyses, ranging from clinical applications, such as biomarker analysis, viral detection, prognostic monitoring and fetal screening, to research applications such as phage-host interactions and intracellular profiling. It can also be applied to assist with the library preparation needed for next-generation sequencing.
4.4.1 Digital PCR Concept
The basic methodology with dPCR is to partition a DNA sample into thousands or tens of thousands of separate reaction chambers so that each contains one or no copies of the sequence of interest. Similar to qPCR, the amplicons are then hybridized with fluorescence probes, which allows the detection of sequence-specific products. PCR is performed in each partition, scoring a positive or negative for the presence of the target sequence based on its fluorescence, and returning an absolute value of DNA concentration.
dPCR improves upon the sensitivity of qPCR and enables the detection of signals by overcoming the difficulties inherent in amplifying rare sequences. The critical step is sample partitioning. By separating each DNA template, this technique, essentially, performs individual amplification reactions identified with fluorescence probes. Unlike qPCR, dPCR does not rely on the number of amplification cycles to determine the initial amount of template DNA in each sample. Rather, it relies on Poisson statistical analysis to determine absolute quantity of template DNA.
4.4.2 Digital PCR Platforms
There are currently two types of digital PCR platforms:
In this SelectScience video interview, Carolyn Reifsnyder, Bio-Rad Laboratories, Inc., introduces new assays for genome edit detection using the droplet digital PCR system, as well as the new ddSEQ™ Single-Cell Isolator, at the AACR conference in 2017.
Caroline Reifsnyder, Bio-Rad Laboratories, Inc., explains how new assays working in conjunction with the droplet digital PCR system enables faster, cost-effective, and precise validation of genome editing events. Watch the video here.4.4.3 Applications of Digital PCR
Digital PCR was originally developed as a technique to investigate rare variants of minority targets such as mutations, while in the presence of large numbers of wild-type sequences. The enhanced sensitivity and dynamic range offered by dPCR have facilitated the use of this method in a multitude of applications. Some examples of applications include copy number variation, rare sequence detection, gene expression and miRNA analysis, single-cell analysis, pathogen detection and next-generation sequencing sample preparation.
In an exclusive SelectScience editorial article, Dr. Bruce Conklin, Gladstone Investigator, and Professor in the Division of Genomic Medicine at University of California, San Francisco, shares his research on rare mutation detection. The Conklin lab has developed a robust, sensitive protocol to detect single nucleotide substitutions using digital PCR.
Next-Generation Sequencing (NGS): Studies have found that dPCR quantification is a more accurate and precise method for quality control of NGS libraries than conventional qPCR methods. NGS library quality control is essential for optimizing sequencing data yield, thereby increasing efficiency and throughput while lowering cost. Predominantly, NGS libraries are quantified using qPCR and their size determined by gel or capillary electrophoresis. However, these techniques have limitations and the steps to rectify them can be time-consuming and expensive. By adopting dPCR technology, it is possible to avoid the high cost of failed runs and suboptimal data yields.
Clinical diagnostics: Traditional PCR used extensively for clinical diagnostic applications suffers from lack of sensitivity. Digital PCR, however, can detect minute changes in gene sequence or quantity; it also provides a definitive, quantitative measurement of nucleic acid. This has potential for many areas of diagnostics, including non-invasive tumor mutation detection from body fluid analysis, and screening for tumor and pathogen resistance.
In this SelectScience video, Emil Christensen from Aarhus University, Denmark, discusses the use of droplet digital PCR (ddPCR) in monitoring the progression of bladder cancer in patients.
Figure 5. Emil Christensen anlyzes liquid biopsies in urine and plasma samples to detect mutations in bladder cancer. Watch the video here.
4.5 Other Variations of PCR
Variations to PCR can be achieved by making small alterations to the PCR protocol and/or the components of the PCR. Some of the most common types include:
Long PCR: In basic PCR, the efficiency of amplification, and therefore the yield of desired amplified products, decreases significantly as the amplicon size increases over 5kb. Amplification of DNA fragments larger than 5kb is desirable for numerous applications, including genome cloning, sequence mapping and gene cluster studies. Long PCR uses a blend of thermostable DNA polymerases and allows for the amplification of longer fragments.
Multiplex PCR: Multiplex PCR involves using multiple sets of primers for the amplification of several targets in a single PCR experiment. It is commonly used for pathogen identification, high-throughput SNP genotyping, mutation and gene deletion analysis, and also in forensic applications for human identification studies.
Colony PCR: Colony PCR is commonly used following transformation to screen colonies for the desired plasmid. In colony PCR, following the addition of a small quantity of cells to the PCR mix, the temperature and length of the initialization and/or denaturation steps are adapted to release the DNA from the cells, which is then available for amplification.
Nested PCR: In nested PCR, a second round of amplification is performed following conventional PCR, using a second set of primers that are specific to a sequence found within the DNA of the initial conventional PCR amplicon. The use of a second amplification step with ‘nested’ primers results in a reduction of nonspecific binding and increases the amount of amplicon produced.
Touchdown PCR: Touchdown PCR (TD-PCR) involves using a cycling program with varying annealing temperatures. In TC-PCR, an annealing temperature that is above the melting temperature of the primer is used for the initial cycle. It is then decreased in increments for subsequent cycles. The primer will anneal at the highest temperature that it is able to tolerate and is least permissive of nonspecific binding. TD-PCR therefore enhances the specificity of the initial primer-template duplex formation and hence the specificity of the final PCR product.
Allele-Specific PCR: Allele-Specific PCR (AS-PCR) is a convenient and reliable method for genotyping SNPs and mutations. In conventional PCR, primers are chosen from an invariant part of the genome and might be used to amplify a polymorphic area between them. In AS-PCR, at least one of the primers is designed from a polymorphic area with the mutation(s) located near its 3’-end. Under stringent conditions, the mismatched primer will not initiate replication, whereas a matched primer will permit amplification. The appearance of an amplification product therefore indicates the genotype.
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