PCR Buying Guide
The polymerase chain reaction (PCR) is a technique used to amplify DNA sequences in vitro and is widely used for many applications. PCR is an extremely broad technique and consequently choosing the right products for your application can be difficult.
This guide provides you with a brief overview of the techniques used in PCR and highlights the technologies that are currently available. You will learn what factors need to be considered when buying kits and reagents for PCR and about the future applications of this technology.
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. In the first step of PCR the sample is heated to between 94°C and 98°C, which initiates melting or denaturation the double-stranded DNA (dsDNA). In the second step, the temperature is decreased to between 50°C and 65°C, to allow the annealing of the primers to specific sequences of the now single stranded DNA. In the third step the temperature of the reaction is increased, typically to 72°C, for primer extension. During this step the DNA polymerase synthesizes new DNA strand complementary to the template DNA strand through the addition of dNTPs in the 5’ to 3’ direction. This three step sequence of denaturation, annealing and extension, is then 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 by running DNA 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.
Other Variations of PCR
Other variations of 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 of PCR are summarized below. It is important to note that many more methods have been developed and new methods are continuously being designed.
- 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 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 set results reduces nonspecific binding in products 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 which is least-permissive of nonspecific binding that it is able to tolerate. 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, where as a matched primer will permit amplification. The appearance of an amplification product therefore indicates the genotype.
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General Considerations for PCR (Reagents, Polymerases, Consumables and Cleanup)
Over the past two decades numerous PCR-based methods have been developed and the type of method used will depend on the application it is being used for. PCR is a very broad technique, and consequently the factors that need to be considered when purchasing products for PCR will be largely dependent on the type of PCR being used. In this section the key considerations that are applicable to the majority of PCR experiments will be discussed. Please visit the SelectScience product library to find out more about the latest PCR products and read user reviews.
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 that 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.
In the absence of a master mix it is important to consider each of the components of the PCR setup. While it may seem trivial, water can be a source of 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 repeated freezing and thawing.
In PCR, 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 failed experimentation.
Successful PCR amplification is largely dependent on the quality and quantity of the template. 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. These impurities, which are commonplace in home-made template preparations, can reduce the sensitivity and efficiency of PCR amplification. 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, on the basis of which, a suitable DNA polymerase can be selected:
- 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.
Many scientists do not give the plasticware they use in their laboratory too much thought, but it is important to note not all plasticware is created equal. Different consumables can make a huge difference in the quality and reproducibility of your 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.
Choosing the appropriate consumables for PCR will be largely dependent on the level of throughput. PCR tubes and their associated caps can be purchased individually or in strips and are ideal for low throughout PCR. PCR plates, which are available in many formats, are designed for high throughput PCR. 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.
When purchasing such consumables for PCR it is important to check whether they are compatible with the PCR instrumentation that is being used in your laboratory. 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 prior 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 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 carrying out the extraction to dangerous chemicals.
Often the simplest, quickest, most convenient and most reproducible approach for PCR cleanup is to use a commercial PCR purification kit. 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 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 downstream applications such as ligations reactions, restriction digests, sequencing and in vitro transcription/translation.
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Considerations for 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. Specialized 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
In real time PCR 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.
Although dsDNA-binding dyes provide the simplest and cheapest option for real time PCR, 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.
During the past decade, several high-profile cases of faulty research have been linked to inconsistent real time PCR techniques and experiments. Until recently there was a lack of consensus regarding how best to perform and interpret quantitative real time PCR experiments. The problem was exacerbated by a lack of sufficient experimental detail in many publications, which impeded the reader's ability to critically evaluate the quality of the results presented or to repeat the experiments.
In April 2009, Stephen Bustin, a molecular science professor at the school of medicine and dentistry at Queen Mary University of London and an international team of nine scientists, joined forces and developed a set of guidelines for the publishing real time PCR results. The resulting 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 not only help to simplify PCR workflows, but also help scientists conform to MIQE guidelines.
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Overcoming Common PCR Challenges (GC-Rich Templates, Crude Samples and Long Range)
Over the past few years several PCR 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. In this section some of the most common challenges in PCR are discussed and the current methods for overcoming such challenges are reviewed.
The efficiency of PCR amplification is influenced by the nucleotide composition and sequence of the template DNA. 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. Despite the inherent challenges, detection of GC-rich sequences is becoming increasingly important and is required for the molecular diagnosis of inherited diseases for example.
Many different methods have been developed to facilitate the amplification of GC-rich templates. 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.
There are many DNA polymerases available that 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 and allows for 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 testing. Blood samples can be used for PCR-based diagnosis of microbial infection, genetic disease, forensic analysis, blood banking and for human DNA identification. 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 are not always efficient and can lead to loss of the target DNA. Furthermore, these procedures are time-consuming and 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. In addition, 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 for many scientists, but as novel enzymes and enhancers continue to be developed, crude samples such as blood, plasma, soil and tissues are likely to be used more frequently in PCR experiments.
Long Range PCR
PCR products of up to 5 kb can usually be amplified using standard protocols. However, 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.
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The Future of PCR
Today, PCR is used in fields as broad as ecology, diagnostics, forensics, and food safety, while at the same time remaining an important tool in molecular biology. PCR methodologies and technologies have evolved dramatically over the years, although the fundamental procedure – denature, anneal, extend – has not changed much. Countless variations on the basic PCR procedure have been developed and applied to answer unique scientific questions. 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 thermocyclers and real time PCR instruments. More recently digital PCR methodologies and associated microfluidic devices for handling high-throughput PCR applications have been developed. For each new technology, method, application and challenge new PCR kits and reagents have been designed.
Digital PCR (dPCR) is a relatively new approach to nucleic acid detection and quantification. In dPCR a sample is diluted and partitioned into hundreds or even millions of separate reaction chambers so that each contains one or no copies of the sequence of interest. As with real time PCR the amplicons are hybridized with fluorescence probes, such as molecular beacons, that allow the detection of sequence-specific products using different fluorophores. By counting the number of 'positive' partitions (in which the sequence is detected) versus 'negative' partitions (in which it is not), it is possible to determine exactly how many copies of a DNA molecule were in the original sample. In dPCR, because the fraction of negative reactions is used to generate an absolute count of the number of target molecules in the sample, there is no need for reference to standards or endogenous controls.
dPCR has many applications, including the detection and quantification of low-level pathogens, rare genetic sequences, copy number variations, and relative gene expression in single cells. dPCR has the potential to be a key molecular tool for both research and applied purposes, revolutionizing fields ranging from tumor mutation analysis and infectious disease diagnosis, to monitoring of genetically modified organisms. When compared with qPCR, dPCR offers a simple method for very sensitive detection of rare mutations and genomic instability.
One of the most important determinants of accuracy in dPCR is the partitioning of the sample. Advances in nanofabrication and microfluidics have now led to systems that produce hundreds to millions of nanoliter- or even picoliter-scale partitions. Some dPCR systems create reaction chambers within specially designed chips or plates, while others sequester reagents into individual droplets.
Although dPCR involves a simpler workflow, and is often considered more accurate, compared to real time PCR, the current technology is quite low throughput. Currently only a handful of companies have commercialized products for dPCR and despite the concept of PCR being around for over two decades, this technique is still considered to be in its infancy. The dPCR market is however predicted to grow to nearly $250 million globally by 2016, changing dPCR from a niche technology to something that is used as a standard tool in the lab. As with PCR methods in the past, incremental steps will be made, with some groups developing new platforms, others optimizing reagents and procedures, and each of these advances eventually coming together to present low input, high-throughput PCR options that are available to researchers in many laboratories as well as those working in field environments.
Given that reproducibility and standardization in each step of a PCR experiment is the basis for successful results, 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. Such demands can only be met through the use of automated PCR systems.
Manual PCR setup is error prone due to pipetting variability, which can result from incorrect pipette calibration and/or human error. This leads to inconsistencies between different experiments, different laboratories and different researchers. Maintaining pipetting precision and ensuring reproducibility is even more difficult when pipetting into multi-well plates. Manual pipetting of PCR reagents increases the risk of contamination with nucleases, and this is especially critical when using RNA as a template. In addition, manual pipetting is time consuming, tedious and can result in repetitive strain injury.
The advantages of automating PCR setup include 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.
Post-PCR analysis and detection is commonly performed using agarose gel electrophoresis. Traditional agarose gel electrophoresis is time consuming and labor intensive, especially if a large number of samples need to be analyzed. Gel preparation also involves exposure to hazardous chemicals such as ethidium bromide. Thorough analysis of data in terms of fragment sizes and concentration is challenging, especially when data are to be compared with previously analyzed PCR products. Several factors such as the agarose quality and the percentage of agarose used affect the duration of electrophoresis and can influence results. Use of a high voltage during an electrophoretic run often results in smearing of nucleic acids, making analysis of results difficult. Standardization is of key importance when comparing data from different gel runs and this places a great emphasis on accurate electrophoresis conditions and record keeping. 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.
When purchasing equipment for the automation of PCR it is important to consider the costs of associated consumables and/or check whether the systems are compatible with multiple consumable brands. It is important to be aware of the technical support and training that is available immediately following purchase and in the long term. Furthermore, it is important to be aware of the potential expandability of the instrument for future PCR applications. In the future PCR automation will continue to evolve as a result of the continued development of new methods and applications.
Multiplex PCR employs different primer pairs in the same reaction for simultaneous amplification of multiple targets. Multiplex PCR 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. For multiplex PCR it is advisable to use commercially available kits with specialized enzymes and optimized buffer systems. This is because compared with standard PCR systems using only 2 primers, an additional challenge of multiplex PCR is the varying hybridization kinetics of different primer pairs. Primers that bind with high efficiency could utilize more of the PCR reaction components, thereby reducing the yield of other PCR products.
As an extension to the practical use of PCR, this technique 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.
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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PCR is routinely used in all areas of science, from molecular biology and clinical diagnostics to forensic science, environmental science and food science. PCR is an extremely broad technique and consequently choosing the right products for your application can be difficult.
Visit the SelectScience PCR product directory for an overview of the latest products from leading manufacturers and read user reviews from other members. Keep up to date with the latest techniques and advances in PCR by visiting the SelectScience application notes and video libraries.
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“Highly reproducible results obtained from this kit.”
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