PCR Technology Buying Guide
The polymerase chain reaction (PCR) is a technique used to amplify DNA sequences in vitro, widely used for many applications such as: molecular biology; microbiology; genetics; diagnostics; clinical laboratories; forensic science; environmental science; food science; hereditary studies and paternity testing.
This buying guide provides you with an overview of the key technologies and considerations in Real Time PCR, Digital PCR and other methods.
1. Introduction to PCR
2. PCR Methods
3. PCR Reagents
4. DNA Polymerases
5. PCR Consumables
6. PCR Clean-up
7. Real-time PCR
8. Overcoming Common PCR Challenges
9. Digital PCR
10. The Future of PCR: Automation and Multiplexing
Introduction to PCR
The Polymerase Chain Reaction (PCR) is an in vitro method used for the amplification of DNA. Since the introduction of PCR in 1985, it has become an indispensable technique used in a broad range of scientific laboratories for a variety of applications. These include cloning, gene expression analysis, genotyping, sequencing and mutagenesis. PCR is also commonly used in disease diagnostics, paternity testing, forensic investigation, food testing and environmental science.
Despite revolutionizing scientific research, the principle of basic PCR is elegantly simple and involves a three-step reaction of thermal denaturation, primer annealing and primer extension. A typical amplification reaction is composed of target DNA, a thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer and magnesium.
The three step sequence of denaturation, annealing and extension is repeated for several cycles, resulting in the exponential amplification of the input DNA. DNA amplification is typically confirmed using agarose gel electrophoresis, and although DNA can be quantified during end point analysis it is generally considered more reliable to do this using real time PCR.
While the basic 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:
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.
Reverse Transcriptase PCR
The DNA polymerases used for basic PCR require a DNA template. There are however numerous instances in which amplification of RNA would be preferred. To apply PCR to the study of RNA, 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. Reverse Transcriptase PCR (RT-PCR) is commonly used for gene expression profiling, inserting eukaryotic genes into prokaryotes, and disease diagnosis.
Quantitative Real Time PCR
In traditional PCR the amplified product, or amplicon, is detected by an end-point analysis where DNA is run on an agarose gel following completion of the reaction. In contrast, real time PCR, which may also be referred to as quantitative real time PCR (qPCR), enables the accumulation of amplified product to be detected and measured as the reaction progresses. Typically real time PCR products are detected using either nonspecific fluorescent dyes, which intercalate with any dsDNA or sequence-specific DNA probes, which are labeled with a fluorescent reporter and are detected after hybridization with complementary DNA.
The main advantage of real time PCR, compared to traditional PCR, is that real time PCR enables the starting template copy number to be determined with accuracy and high sensitivity over a wide dynamic range. Frequently real time PCR is combined with RT-PCR to quantify messenger RNA, microRNA and non-coding RNA. 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.
In its simplest form, digital PCR (dPCR) is a single-molecule counting method that gives a direct quantitative measurement of absolute DNA concentration and thus does not require a standard curve. The main principle of digital PCR is that a single sample is split into many fractions, all of which are subsequently analyzed by a standard PCR method. By isolating individual DNA templates, this process effectively enriches DNA that was present at very low levels against background noise. Since the data is recorded as either positive or negative, the results are considered qualitative, hence digital in nature.
Other Variations of PCR
Other variations 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. Read more in the ‘Future of PCR’ Section.
- 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 step 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, and is then decreased in increments for subsequent cycles. The primer will anneal at the highest temperature that it is able to tolerate and 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.
Despite the numerous variations on the basic theme of PCR, the reaction itself is composed of only a few components. These are target DNA, DNA polymerase, two oligonucleotide primers, dNTPs, water, reaction buffer and magnesium. Many scientists choose to use PCR master mixes, as these simplify the reaction setup, save time, enable consistency and reduce the risk of contamination.
PCR master mixes provide the key ingredients necessary for performing PCR in a premixed and optimized format, which streamlines and simplifies the PCR workflow. Master mixes come pre-measured and contain the DNA polymerase, salts, magnesium, dNTPs and optimized reaction buffer. Typically PCR master mixes are supplied as at least a 2X concentrate and the DNA template and primers are the only additions necessary to perform PCR. Choosing the right PCR master mix will be dependent on the type of PCR being performed and the application. For example, different master mixes are available for routine PCR, hot start PCR, high fidelity PCR and real time PCR. Additionally, different PCR master mixes may contain PCR enhancing additives, dyes and/or genetically optimized polymerases. Understanding the different types of DNA polymerases available for PCR and the different detection methods available for real time PCR will be important in choosing a suitable master mix. These will be discussed in detail shortly.
The water source can be a concern and frustration in PCR experiments. In addition to being nuclease-free to prevent nucleic acid degradation, water for PCR experiments should also be free of specific ions, organics and bacteria. High quality laboratory purified water may be sufficient for PCR, but in the absence of a suitable water purification system, it is recommended that ultrapure bottled water is used.
The PCR reaction buffer is responsible for providing an environment with an appropriate pH and ionic strength for optimal DNA polymerase activity. Due to the differential requirements of various DNA polymerases, reaction buffers are typically supplied with the DNA polymerase and most often as a 10X concentrate. dNTP’s can be purchased as a premixed solution of dATP, dTTP, dCTP, and dGTP, or they can be supplied individually. The quality of dNTP’s is vital to the success of PCR experiments and it is recommended that once purchased dNPTs are aliquoted into smaller volumes, as they can be damaged by repeated freezing and thawing.
Magnesium is required as a cofactor for thermostable DNA polymerases. In the absence of adequate free magnesium, DNA polymerases will be inactive. Conversely, if the concentration of magnesium is too high the fidelity of DNA polymerases will be reduced and nonspecific amplification may occur. Many reaction buffers that are supplied alongside the DNA polymerase already contain magnesium chloride (MgCl2). It is recommended that scientists empirically determine the optimal magnesium concentration for each target, thus magnesium-free reaction buffers are often preferred. When choosing DNA polymerases it is also therefore important to consider whether the supplied reaction buffer is also suitable for your PCR needs.
Good primer design is essential for successful PCR. Considerations will include primer length, melting temperature, specificity, GC content, secondary structure and 3’-end stability. A number of primer design tools are available that can assist in PCR primer design for new and experienced users alike. These tools may reduce the cost and time involved in experimentation by lowering the chances of failure.
The PCR template quality and quantity often determines whether PCR amplification is successful. Because PCR consists of multiple cycles of enzymatic reactions, it is more sensitive to impurities such as proteins, phenol, chloroform, salts, ethanol, EDTA, and other chemical solvents than single-step, enzyme-catalyzed processes. There are numerous PCR sample preparation kits available to purify nucleic acids from a wide variety of sample materials, such as whole blood, bacterial cells, viruses and tissues. The amount of template in a reaction also strongly influences performance in PCR. The concentration of template required is dependent on the complexity of the sample and the PCR method. Plasmid DNA, for example, is small and highly enriched for the specific target sequence, while genomic DNA usually contains only one copy of the target sequence per genome equivalent. Thus it is necessary to use more of the latter than the former to present a sufficient number of targets for efficient amplification. Typically recommended amounts for standard PCR are <500 ng for genomic DNA (human), between 1 and 10 ng for bacterial DNA and between 0.1 and 1 ng for plasmid DNA.
Prior to the use of thermostable DNA polymerases in PCR, researchers had to laboriously replenish the reaction with fresh enzyme, such as Klenow or T4 DNA polymerase, after each denaturation cycle. Thermostable DNA polymerases revolutionized and popularized PCR because of their ability to withstand high denaturation temperatures. The use of thermostable DNA polymerases also allowed higher annealing temperatures, which improved the stringency of primer annealing.
Selecting an appropriate thermostable DNA polymerase, in accordance to the application, is extremely significant for the success of a PCR experiment. Choosing an appropriate enzyme can be challenging as there are a vast number of commercially available enzymes to choose from. DNA polymerases possess the following properties:
- Thermostability: Stability can be measured in terms of how long the enzyme retains at least one-half of its activity during sustained exposure to high temperature. For PCR experiments it is imperative that the DNA polymerase retains its activity following template denaturation.
- Elongation Rate: The number of nucleotides added per second per molecule of DNA polymerase is known as the elongation rate. Generally, higher rates of elongation are desired as this facilitates faster DNA extension, higher DNA yields and shorter cycling times.
- Processivity: The average number of nucleotides added by a DNA polymerase enzyme per association/dissociation with the template is referred to as the processivity. The processivity of the DNA polymerase influences the overall speed and yield of the PCR reaction.
- Fidelity: DNA polymerase fidelity or specificity refers to the ability of a polymerase to select a correct dNTP from a pool of structurally similar molecules. High fidelity DNA amplification is best achieved by using a polymerase with proofreading activity.
There are some important differences between proofreading and non-proofreading DNA polymerases. Firstly, proofreading DNA polymerases are more accurate than non-proofreading DNA polymerases due to having 3’ to 5’ exonuclease activity, which can remove a misincorporated nucleotide from a growing DNA chain. Secondly, amplification with non-proofreading DNA polymerases results in the template-independent addition of a single nucleotide to the 3’-end of the PCR product, whereas proofreading DNA polymerases remove the unpaired 3’ overhanging nucleotides to create blunt ends. Typically when the amplified product is to be cloned, expressed or used in mutation analysis a proofreading DNA polymerase is a better choice due to its high fidelity. However, for routine PCR, where simple detection of an amplicon is the goal, non-proofreading DNA polymerases are commonly used as the yields tend to be higher with these enzymes.
Several types of DNA polymerase with different properties for various applications are now available. Taq DNA polymerase, a highly thermostable DNA polymerase from the thermophilic bacterium Thermus aquaticus, has become the standard reagent for routine PCR. Taq DNA polymerase is commercially available in both the native form and a cloned version that is expressed in non-thermophilic host bacteria. Taq DNA polymerase generates products with an adenosine overhang at the 3’-end, thus should the fragment be amplified for cloning purposes, a T/A cloning vector would be required. This enzyme possesses maximum catalytic activity between 75°C and 80°C and has a half-life of approximately 40 minutes at 95°C. Taq DNA polymerase exhibits good processivity and has an elongation rate of >60 nucleotides per second at 75°C, which means that extension times of 1 minute per 1kb of DNA template to be amplified should be sufficient. While Taq DNA polymerase is suitable for many PCR applications, such as colony PCR and routine amplification of DNA fragments up to 5kb, it is not suitable for applications where high accuracy is desired. One of the main limitations of Taq DNA polymerase is its low replication fidelity, which is a result of the enzyme lacking 3’ to 5’ exonuclease proofreading activity. The error rate of Taq DNA polymerase is among the highest of the thermophilic polymerases at between 1 x 10-4 and 2 x 10-5 errors per base pair.
Tfl DNA polymerase and Tth DNA polymerase are often considered similar to Taq DNA polymerase in that they lack 3’ to 5’ exonuclease activity and are suitable for many routine PCR applications. Tfl DNA polymerase is suitable for applications requiring high temperature synthesis of DNA, such as high temperature DNA sequencing. While Tth DNA polymerase is suitable for routine PCR in the presence of Mg2+ ions, it also has an intrinsic reverse transcriptase activity in the presence of Mn2+ ions, thus can be used for both PCR and RT-PCR.
Pfu DNA polymerase, an enzyme from the hyperthermophilic archaeum Pyrococcus furiosus, is often the enzyme of choice for high fidelity PCR. Pfu DNA polymerase exhibits the lowest error rate of any thermostable DNA polymerase studied, which is attributable to its high 3’ to 5’ exonuclease proofreading activity. This makes Pfu DNA polymerase appropriate for applications such as gene cloning, gene expression and mutation analysis. Other proofreading enzymes commonly used in high fidelity PCR include Tli DNA polymerase, which is also known as Vent polymerase, Deep Vent DNA polymerase and KOD DNA polymerase. These enzymes typically exhibit 5- to 15-fold higher fidelity than Taq¬ polymerase, but are considerably slower, have lower processivity and require significant optimization.
Many commercially available DNA polymerases have been modified to enhance their performance in a given application, or to overcome specific challenges. In hot start PCR, for example, specialized DNA polymerases can be used to suppress nonspecific product amplification at low temperatures and increase the overall yield of the desired product. This is most commonly achieved through the use of blocking antibodies, which will dissociate from the DNA polymerase at high temperatures, or through chemical modification that renders the enzyme inactive until higher temperatures are reached. Many other commercially available DNA polymerases have been specifically engineered to overcome the shortcomings of existing polymerases and enhance their processivity, extension rates, stability and/or fidelity. For example, DNA polymerases are commonly fused with a DNA binding domain to create a chimeric enzyme that has enhanced processivity. More nucleotides are added per DNA polymerase binding event, thus the extension time required is shorter and the yield is also improved.
Once the appropriate enzyme has been selected it is important to follow the manufacturer’s instructions with regard to enzyme concentration. DNA polymerases from different suppliers may behave differently because of different formulations, assay conditions, and/or unit definitions. Additionally, the concentration of enzyme required will depend on the template, primers and cycling conditions. It is common to have the enzyme slightly in excess so the reaction can proceed efficiently. Adding too much enzyme however, can be detrimental to the experiment and result in the accumulation of nonspecific background products. The enzyme concentration can be optimized for PCR by testing a range of different concentrations, typically from 0.5 to 5 units/100 µL.
It is important to note not all plasticware is created equal. Different consumables can make a huge difference in the quality and reproducibility of PCR results. Specialized tubes, plates, caps, seals and pipette tips have been designed specifically for optimal PCR performance.
PCR consumables, such as reaction tubes and plates, need to be mechanically stable in order to withstand high temperatures, but have thin enough walls to permit efficient heat transfer. All PCR consumables need to be free of contamination. In addition, for many applications the color of the plastic is also an important consideration. For example, white-wells are often preferred in tubes and plates for real time PCR as they reflect fluorescence and reduce cross-talk to provide a better signal to noise ratio compared to clear or frosted wells.
When purchasing such consumables consider compatibility with your PCR instrumentation and the level of your throughput. PCR tubes purchased individually or in strips, are ideal for low throughput PCR, whereas PCR plates are ideal for high throughput. PCR plates often need to be sealed to prevent evaporation during PCR, reduce cross-contamination and/or enable efficient transport and storage. Plate sealing is commonly achieved using either adhesive, or heat sealing films and foils.
Good laboratory practice is essential for successful PCR regardless of quality of your consumables, reagents and water. It is advisable to create pre- and post-amplification work areas in your laboratory to minimize cross-contamination between samples and prevent carryover of RNA and DNA from one experiment to the next. Gloves should be worn throughout PCR experiments and changed regularly. In addition to good laboratory practice several methods have been developed to help prevent contamination including the use of isopsoralen and uracil-N-glycosylase (UNG).
For many downstream applications it is necessary to remove unincorporated primers, excess dNTPs, proteins and salts from the amplification product following PCR. Several different methods have been developed for PCR cleanup and the choice of method will largely depend on the number and type of PCR samples to be purified and the downstream application.
Many scientists choose a simple ethanol precipitation method for DNA isolation. While this technique removes most of the dNTPs and salts from the sample, the proteins and primers are often left behind. Phenol-Chloroform extraction is a liquid-liquid extraction technique that is also commonly used for nucleic acid isolation. The phenol-chloroform technique is a popular method as it is relatively cheap and can be used on a wide range of sample types. It is particularly effective for extracting large amounts of high molecular weight DNA. However, this method does also have some disadvantages including being very labor intensive, being easily contaminated and exposing the scientist to potentially dangerous chemicals.
PCR purification kits are often the simplest, quickest, most convenient and most reproducible approach. Most of these kits are based on the retention of DNA fragments greater than around 100 base pairs on some form of solid support, such as a silica membrane. Following washing steps to remove dNTPs, buffer and unincorporated primers, a final elution step allows recovery of the bound DNA in a reasonably small volume. Many commercial kits are based on spin-column technology and can be used for either gel extraction or direct purification of PCR products. For gel extraction, the PCR products are size-fractionated on an agarose gel and the DNA band of interest is then cut from the gel in the smallest volume of agarose, under UV illumination. The gel slice is then melted in the presence of a chaotropic salt, such as sodium iodide, before being absorbed to a membrane in a spin column. The same procedure is used for post-PCR cleanup without gel separation. Gel extraction is useful for samples that require nonspecific amplification products to be separated from the desired product. Typically, PCR cleanup using a kit takes approximately 10 minutes and results in highly purified DNA for use in downstream applications such as ligations reactions, restriction digests, sequencing and in vitro transcription/translation.
Real Time PCR
Real time PCR and RT-PCR enable accurate quantification of starting amounts of DNA, cDNA, and RNA targets. Real time detection of PCR products is made possible by including in the reaction a fluorescent molecule that reports an increase in the amount of DNA with a proportional increase in fluorescent signal. Because it detects the amount of product as the reaction progresses, real time PCR provides a wide linear dynamic range, demonstrates high sensitivity, and is very quantitative.
Real Time Thermal Cyclers Specialized Real time PCR thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence as amplification occurs. When purchasing a new real time PCR instrument, considerations should include the footprint, multiplexing capabilities, compatibility with standard liquid handling systems and multichannel pipettes, reagent and consumables cost, ease-of-use, software capabilities and throughput potential. In addition, it is important to check the level of after sales service and technical support that is available.
In real time PCR, products can be detected using either fluorescent DNA binding dyes that bind to dsDNA or fluorescently labeled sequence-specific probes.
DNA Binding Dyes DNA binding dyes bind reversibly, but tightly to DNA by intercalation and/or minor groove binding. Fluorescent dyes are nonspecific and bind to any dsDNA generated during amplification resulting in the emission of enhanced fluorescence. This allows the initial DNA concentration to be determined with reference to a standard sample. Fluorescence measurements are taken at the end of the elongation step of each PCR cycle to allow measurement of DNA in each cycle.
Although several fluorescent dyes are commercially available, 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. The excitation and emission maxima of SYBR Green are at 494 nm and 521 nm, respectively, and these dyes are compatible for use with any real time cycler. It is likely that when using SYBR Green the concentration will need to be optimized, as SYBR Green is known to inhibit PCR at higher concentrations. Conversely, too low a concentration of SYBR Green may make it difficult to detect the increase in PCR products. 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 setup to examine multiple genes in a real time PCR assay with SYBR Green.
dsDNA-binding dyes provide the simplest and cheapest option for real time PCR, but the principal drawback is that both specific and nonspecific 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 nonspecific PCR products. After completion of the amplification reaction, a melt curve is generated by increasing the temperature in small increments and monitoring the fluorescent signal at each step. As the dsDNA in the reaction denatures (i.e., as the DNA “melts”), the fluorescence decreases. The negative first derivative of the change in fluorescence is plotted as a function of temperature. A characteristic peak at the amplicon’s melting temperature (Tm, the temperature at which 50% of the base pairs of a DNA duplex are separated) distinguishes it from other products such as primer-dimers, which melt at different temperatures.
Fluorescently Labeled Probes Fluorescently labeled probes provide a highly sensitive and specific method of detection, as only the desired PCR product is detected. Several probes based on different chemistries are available for real time detection, these include:
- TaqMan® Probes: TaqMan® hydrolysis probes are sequence-specific oligonucleotide probes carrying a fluorophore and a quencher dye. The fluorophore is attached at the 5' end of the probe and the quencher dye is located at the 3' end. During the combined annealing/extension phase of PCR, the probe is cleaved if the DNA polymerase possesses 5' to 3' exonuclease activity, separating the fluorophore and the quencher dyes. Taq DNA polymerase, Tth DNA polymerase and Tfl DNA polymerase all possess 5’ to 3’ exonuclease activity. The separation of fluorophore and quencher dye results in detectable fluorescence that is proportional to the amount of accumulated PCR product. TaqMan® probes are the most commonly used probes in real time PCR. Different manufacturers use different fluorophore-quencher pairs.
- 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 so that the ends are complementary. When the probe is in solution, the two ends of the probe hybridize and form a stem-loop (or hairpin) structure, with the fluorophore and quencher in close enough proximity so that 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 PCR and are particularly useful for allelic discrimination experiment. Molecular beacons can however be difficult to design. The stem of the hairpin must be strong enough that the molecule will not spontaneously fold into non-hairpin conformations that result in unintended fluorescence. At the same time, the stem of the hairpin must not be too strong, or the beacon may not properly hybridize to the target.
- FRET Hybridization Probes: PCR with fluorescence resonance energy transfer (FRET) probes uses two labeled oligonucleotide probes that are designed to bind to the 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 the emission spectrum of the donor dye overlaps significantly with the excitation spectrum of the acceptor dye, while the emission spectrum of the donor dye is spectrally separated from the emission spectrum of the acceptor dye. 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 is detected during the annealing phase of PCR and is proportional to the amount of PCR product.
Modern Real time PCR platforms typically have multiple detection channels enabling flexibility in the choice of probe labels. 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. In addition, it is important to select quenchers that are compatible with the fluorophore that has been selected. For optimal performance, the quencher’s absorbance spectrum should match the fluorophores emission spectrum as closely as possible. Probe design can be extremely challenging, but many manufacturers provide assistance in this process.
In the simplest real time PCR experiments, one target is amplified and analyzed. However, multiple targets can be simultaneously analyzed through the use of primers or probes labeled with multiple fluorophores. Real time PCR instruments can distinguish and monitor the fluorescence emission spectra of each fluorophore, resulting in quantitation of each target. In multiplex PCR fluorophores with minimally overlapping emission spectra should be selected. For some real time PCR approaches, multiplexing can also be accomplished by amplifying products with distinct melting temperatures.
Kits Optimized for MIQE Guidelines 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 describe the minimum information necessary for evaluating real time PCR experiments and help promote better experimental practice. Kits that have been optimized for various real time PCR experiments help to simplify PCR workflows and help scientists conform to MIQE guidelines.
Overcoming Common PCR Challenges
Over the past few years several kits, reagents and enzymes have been developed to help overcome specific challenges in PCR including those associated with GC-rich templates, crude samples and large DNA targets.
PCR with GC-rich templates (>60%) can be extremely difficult: Localized regions of templates rich in GC residues tend to fold into complex secondary structures that may not melt during the annealing phase of the PCR cycle. These secondary structures cause DNA polymerases to stall, resulting in incomplete and nonspecific amplification. In addition, the primers used to amplify GC-rich regions often have a high capacity to form self- and cross-dimers and have a strong tendency to fold into stem-loop structures that can impede the progress of the DNA polymerase along the template molecule. Amplification of full-length GC-rich template DNA is often inefficient, and the products of the reaction can contain a high proportion of shorter molecules that result from blockage of the DNA polymerase. Detection of GC-rich sequences is becoming increasingly important, for example it is required for the molecular diagnosis of inherited diseases.
Several strategies involve the addition of specific reagents to the reaction mix. Betaine (0.8-1.6 M) is the most commonly used additive for enhancing the amplification of GC rich sequences and is used to reduce the formation of secondary structures in GC-rich regions. Alternatively, DMSO (3-20%) and formamide (5-20%) are thought to aid the amplification of GC-rich templates by interfering with hydrogen bond formation between two DNA strands. Good results have also been obtained through the addition of glycerol or tetramethylammonium chloride (0.01-10 mM) to the reaction mix. These enhancing reagents are often used in combination. Several optimization kits incorporating these and other enhancing agents, and a variety of buffers, are currently marketed.
DNA polymerases are available which have been engineered specifically for the amplification of GC-rich DNA. The unique properties of such enzymes may include enhanced processivity and improved tolerance to DNA melting agents. These enzymes are usually supplied with buffers that have also been optimized for GC-rich DNA targets. Another approach that has been developed to enable amplification of sequences high in GC content involves using modified dNTPs. One of the most noteworthy modified dNTPs used to amplify GC-rich targets is 7-deaza-dGTP, a dGTP analog that lacks nitrogen at the 7 position of the purine ring. The use of this modified nucleotide blocks Hoogsteen bond formation without interfering with normal Watson-Crick base pairing, reducing the probability that secondary structures, such as G quadruplexes, will form.
Crude Sample PCR
The ability to use crude samples as templates in PCR eliminates the need for laborious and costly DNA extraction procedures, enabling faster turnaround times from sampling to results. The feasibility of high-throughput or routine crude sample PCR has remained low due to the fact that wild-type DNA polymerases are easily inhibited by several components of crude samples, yielding low success rates and inconsistent results.
PCR-based tests of blood and soil samples are currently used for diagnostics, forensic analyses and environmental applications. Several major inhibitors of PCR in human blood have been reported including hemoglobin, IgG, lactoferrin, bile salts and several anticoagulants. Sensitive and precise PCR detection of microorganisms in soil is necessary for agricultural purposes, infectious disease control and bioterrorism-related pathogen tests. The most potent inhibitor for soil-based PCR is humic acid. The inhibitory effect of blood and soil on PCR is associated primarily with inactivation of the DNA polymerase and/or capturing or degradation of the target DNA and primers.
Several methods have been developed to remove PCR inhibitors from crude samples. However, these various purification procedures can be inefficient and can lead to loss of the target DNA. These procedures are time-consuming, labor-intensive and increase cost. In addition, the multiple sample manipulations involved increase the risk of cross-contaminations. Some PCR enhancers have shown to be effective in relieving polymerase inhibition and enhancing amplification. Several novel DNA polymerases engineered specifically for crude sample PCR have now been developed and are commercially available. Currently, DNA extraction prior to PCR appears to be the preferred method of choice.
Long Range PCR
Amplification of products longer than 5 kb often fails without lengthy optimization. Reasons for failure may include nonspecific primer annealing, secondary structures in the DNA template, and suboptimal cycling conditions. To overcome the challenges of long DNA templates many manufacturers supply optimized systems containing an enzyme blend. Typically these will contain Taq DNA polymerase and a thermostable high fidelity DNA polymerase with proofreading activity. The 3' to 5' proofreading exonuclease activity removes misincorporated bases, allowing subsequent product extension to proceed quickly and efficiently, making amplification of long DNA fragments possible. Thus, the two enzymes act synergistically to generate long PCR products with greater yield and fidelity than Taq DNA polymerase alone.
There are many different kits available for long range PCR and it is important to choose a kit that has been optimized for both your template size and template type. PCR kits are now available that enable the amplification of DNA >30 kb.
Advances in PCR technology have transformed non-quantitative methods of PCR and real-time amplification measurements, but most recently developments have extended to absolute quantitation in the form of digital PCR (dPCR). Digital PCR is a technology which provides absolute counts of target DNA, as well as increased sensitivity, precision and reproducibility compared to the aforementioned qPCR.
There is growing interest in digital PCR because technological progress has continued to make it a more practical and increasingly affordable technology. There are reports stating that nearly a third of researchers queried were looking to add dPCR tools to their labs in the near-future1 2. Scientists have long relied on their gold-standard qPCR to quickly and accurately detect and quantify target DNA, but the future implications of dPCR are challenging this reliance.
Over time the hurdles preventing the widespread adoption of dPCR are being reduced, such as price and more recently the creation of dMIQE guidelines3 (Minimum Information for Publication of Quantitative Digital PCR Experiments). The increasing impact of dPCR on research and clinical diagnostic applications has prompted the development of a robust set of guidelines for scientists. The rapid and universal adoption of the dMIQE guidelines should result in more reproducible data and reliable scientific reporting by standardizing experimental protocols, therefore increasing the impact of the associated research and maximizing the contributions of dPCR within the scientific community.
Digital PCR is progressing from a method that is limited by technical complexity toward a mainstream technology that has unique advantages and applications. It 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. Digital PCR can also be applied to assist with the library preparation needed for massively parallel next generational sequencing methods.
Methodology and Platforms
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. Similarly to qPCR, the amplicons are then hybridized with fluorescence probes, which allow 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 their fluorescence, and returning an absolute value of DNA concentration.
In conventional qPCR, the signal from wild-type sequences can dominate and obscure the signal from rare mutants. Digital PCR improves upon the sensitivity of qPCR and enables the detection of these signals by overcoming the difficulties inherent in amplifying rare sequences. The critical step is sample partitioning. By separating each DNA template, essentially individual amplification reactions are conducted and identified with fluorescence probes. Unlike qPCR, digital PCR 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. Thereby nullifying the need for reference to standard curves or endogenous controls.
Originally digital PCR could be performed manually, but was considered labor-intensive and error prone, in addition to limiting replication levels. Fortunately the technology has advanced tremendously and today’s scientist has several options at their disposal.
There are currently two types of Digital PCR platforms:
- Droplet Digital PCR (ddPCR) utilizes microfluidics to emulsify samples in oil, creating reproducible droplets to be processed and analyzed by fluorescence. This technology has been pioneered by Bio-Rad Laboratories and RainDance Technologies, which differ in their implementation by creating individual nanoliter or picoliter droplets respectively.
- A platform based on integrated fluidic circuits (chips), sometimes abbreviated to qdPCR. Chip-based techniques have a narrower dynamic range, however they provide more precise sample partitioning that can provide consistently high data collection with vastly reduced variance. The companies most committed to this methodology include Life Technology and Fluidigm.
Benefits of Digital PCR
The latest ddPCR procedures surpass the performance of earlier digital PCR techniques by resolving the previous lack of scalable and practical implementations. Massively partitioning samples in the fluid phase addresses these concerns, leading to the creation of thousands of droplets means that a single sample can generate extensive amounts of data, which can be supported by statistical analysis.
Additional benefits include unparalleled precision and increased signal-to-noise ratio because high-copy templates and background are diluted, effectively enriching template concentration in target-positive partitions, allowing for the sensitive detection of rare targets and a ±10% precision in quantification.
Furthermore there are economic benefits to be considered, such as reduced consumable costs due to pico- to nanoliter ranges and lower equipment costs thanks to the emulsion-based reaction system, meaning that the PCR reactions can be performed in a standard thermal cycler without complex chips or microfluidics.
Applications and Considerations
Digital PCR was originally developed as a technique to investigate rare variants of minority targets such as mutations whilst in the presence of large numbers of wild-type sequences. As outlined above, dPCR advantages such as greatly enhanced sensitivity and dynamic range have allowed this useful tool to expand into a multitude of applications. Examples include: copy number variation, rare sequence detection, gene expression and miRNA analysis, single cell analysis, pathogen detection and next-generation sequencing sample preparation4.
Next Generation Sequencing (NGS) is an important application. 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 is used extensively for clinical diagnostic applications; however the technique suffers from lack of sensitivity. It was for diagnostic applications that cancer research pioneers Kenneth Kinzler and Bert Vogelstein of Johns Hopkins University developed the concept of dPCR. In 1999 they modified the traditional PCR method to improve its sensitivity, enabling them to quantify KRAS mutations in stool DNA from colorectal cancer patients3.
Digital PCR 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, as well as screening for tumor and pathogen resistance. Another area of potential is for non-invasive pre-natal screening for foetal chromosomal abnormalities5 using cell-free fetal DNA analysis, where fetal DNA usually makes up less than 5% of the DNA in a sample. Additional future applications of dPCR might include super infection analysis, fetal sexing and therapeutic RNA detection.
Digital PCR Summary
A well-designed and optimized dPCR experiment yields highly reproducible and robust results. However before running an experiment, it is important to understand the goal or expected outcomes. Many of the considerations discussed in the qPCR section of this buying guide will apply to dPCR, but suffice to say the principles of target amplification remain the same despite the quantities of reactant. Digital PCR assays focus on the end-point analysis of each partition to generate quantitative data. Therefore, the results depend less on the efficiency of the amplification compared to qPCR.
The presiding dPCR manufacturers have invested heavily into the technique and the market is approaching an inflection point at which implementing dPCR daily has become affordable and practical. The dPCR market is predicted to grow to nearly $250 million globally by 2016 1 2, changing dPCR from a niche technology to something that is used as a standard tool in the lab. If your research can benefit from this technology now may be a good time to invest.
The Future of PCR
Among the most notable advances in PCR in the past 20 years are the discovery and engineering of thermostable polymerases and the construction of automated thermal cyclers, real time PCR instruments and digital PCR methods.
As projects become larger and more laboratories adopt PCR-based tests, consistency, speed and performance become increasingly important. Reliable tools for automated setup of reactions and sensitive platforms for fragment detection are a welcomed addition for many laboratories. Furthermore many laboratories are seeking new tools which enable higher throughput PCR at an increased speed, with smaller reaction volumes and lower costs.
Manual PCR setup can be prone to pipetting variability, which can result from incorrect pipette calibration and/or human error, as well as increased risk of contamination with nucleases (especially critical when using RNA as a template). In addition, manual pipetting may be time consuming, tedious and can result in repetitive strain injury. However please note that many pipette manufacturers have developed products aimed at overcoming of the majority of these issues.
Automating PCR setup has several advantages: better reproducibility; reduced human error artifacts; reduced costs and reduced labor. Such benefits are useful in all laboratories, but especially important in molecular diagnostics and forensic analyses. There are numerous instruments available for automated PCR setup. In the future these will need to be further developed to cater for higher throughput PCR and multiplex PCR, for example. Consider what sections of your workflow you are interested in automating, what level of operator intervention you require and the timeline for your project.
Agarose gel electrophoresis is commonly used for Post-PCR analysis and detection. Several systems are now available for automated DNA fragment analysis. Such instruments eliminate the need for tedious gel preparation, provide greater confidence in data interpretation and reduce manual handling errors.
Other considerations in automation:
- Consumables should be discussed with the automation suppliers.
- Technical support and training: both immediate and ongoing.
- Future expansion requirements: the key attributes when considering the long-term viability of automation are hardware compatibility, application flexibility and an open reagent platform. As your lab continues to develop you will need automation that can meet your future needs, so be sure to purchase a system which can expand to perform additional applications. Furthermore, a flexible platform that can work with multiple vendor PCR reagent kits can protect your lab from external factors such as kit discontinuation.
Multiplex PCR has the potential to produce considerable savings in time, cost and effort within the laboratory, without compromising on the utility of the experiment. Multiplex PCR ensures standardization in certain experiments because identical reaction conditions and template amounts are used, pipetting and cycling condition variations are eliminated, and reliable comparison of results from a large number of fragments is achieved. Multiplex PCR, if sensitive enough, can be used to amplify and detect a single copy of a nucleic acid sequence and may also be used for both end-point and real time PCR applications. Currently applications of multiplex PCR include pathogen identification, high throughput SNP genotyping, mutation analysis, gene deletion analysis, linkage analysis and human identification studies.
It is a challenging application that typically requires more optimization than standard, single amplicon PCR assays. The key to successful multiplex PCR is the ability to define a single set of reaction parameters (reagent concentrations and cycling parameters) that allows for all primers to anneal with high specificity to their target sequences and be extended with the same efficiency. Primer design, enzyme type and buffer composition are critical factors in this challenge. It is advisable to use commercially available kits with specialized enzymes and optimized buffer systems.
It is also important to utilize a unique reporter dye for each target that you want to identify. Manufacturers can provide the relevant information regarding detectable emission wavelengths, so that you can determine which dyes your instrument is capable of detecting once calibrated. It is important to select dyes with little to no overlap in their emission spectra.
The greatest potential for multiplex PCR is the acceleration of the number of reactions that can be carried out simultaneously. Multiplex PCR assays have the potential to become routinely used in diagnostic, food, forensic and research laboratories. However, certain challenges need to be addressed before the full possibilities of multiplex PCR can be explored. For example, exploiting the full potential of multiplex PCR in the future would involve maximizing the number of regions that may be simultaneously amplified. This is especially useful for studying genetic diseases associated with loci without apparent mutation hot spots or those harboring new or previously unidentified mutations.
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1 Market Research. (2012). U.S. qPCR and dPCR Instrumentation Markets. Frost & Sullivan.
2 Tonya Fowler. (2012). Global Quantitative and dPCR Instrumentation and Consumables Purchasing Trends. Frost & Sullivan.
3 Huggett, Jim F.; Foy, Carole A.; Benes, Vladimir.; et al. (2013). The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments. Clinical Chemistry. 59 (6), 892-902.
4 Baker, Monya. (2012). Digital PCR hits its stride. Nature Methods. (9), 541–544.
5 Dennis Lo, Y. M.; Chiu, Rossa W. K.; et al. (2007). Digital PCR for the molecular detection of fetal chromosomal aneuploidy. Proc. Natl. Acad. Sci. USA. 104 (32), 13116–13121.
“Great product. Technical support has been outstanding. Easy to use and straight forward…"
Nelson Ho, University of Guelph
“This machine has lots of extra features. Altogether, it's a good machine for routine PCR reactions…”
Melanie Eckersley-Maslin, Cold Spring Harbor
"We have been using this equipment for 3 years in our lab. It is very easy to use, with simple program set-up for normal PCR reactions."
Divya Kesanakurti, University of Illinois