PCR Technology Buying Guide

PCR image

The polymerase chain reaction (PCR) is a technique used to amplify DNA sequences in vitro. It is widely used for many applications such as molecular biology, microbiology, genetics, pharmaceutical research, 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.

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 basic research, 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.

PCR Methods & Considerations

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

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.

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 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.

BUYING GUIDE TIP: Consider whether you need the thermal cycler to have multiple modes and check the compatibility with the other instrumentation you use in your lab.

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, for example SsoAdvanced SYBR Green Supermix (172–5260), from Bio-Rad, is by far the most widely used dsDNA-specific dye for real-time PCR. SYBR Green binds all dsDNA molecules, emitting a fluorescent signal of a defined wavelength on binding. DNA binding dyes cannot be used for multiplex reactions because fluorescent signals from different amplicons cannot be distinguished from one another. Parallel reactions can, however, be setup to examine multiple genes in a real-time PCR assay with SYBR Green.

Using dsDNA-binding dyes will provide the simplest and cheapest option for real-time PCR, but the principal drawback is that both specific and 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.

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. TaqMan® probes are the most commonly used probes in real-time PCR.
  • 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.
  • 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 RT PCR platforms typically have multiple detection channels, enabling flexibility in the choice of probe labels.

BUYING GUIDE TIP: It is important to select fluorescent labels that are compatible with the detection channels and filters of the real-time PCR instrument you are using.

BUYING GUIDE TIP: Select quenchers that are compatible with the fluorophore selected. For optimal performance, the quencher’s absorbance spectrum should match the fluorophores emission spectrum as closely as possible.

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.

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 describes the minimum information necessary for evaluating real-time PCR experiments, and helps to promote better experimental practice. A practical guide to publishing data that conform to the MIQE guidelines can be read here.

Applications and Considerations of qRT-PCR

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.

High-throughput real-time PCR data collection is becoming increasingly important because screening a large number of samples, especially in the pharmaceutical industry, is often needed to obtain meaningful results. In this application note, an automated liquid handling solution is demonstrated for the generation of qPCR data, from Bio-Rad’s CFX384 Touch Real-Time PCR Detection System.

In this food contamination study, a probe based qPCR assay was developed for detecting toxin-producing molds in food samples, using Agilent’s Mx3005P QPCR instrument.

Mx3005P-qPCR

Figure 1:The Mx3005P qPCR System from Agilent

Digital PCR

Digital PCR is a technology that provides absolute counts of target DNA, as well as increased sensitivity, precision and reproducibility, compared to qPCR. Digital PCR is progressing from a method that is limited by technical complexity towards 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 generation 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.

Digital PCR improves upon the sensitivity of qPCR and enables the detection of signals by overcoming the difficulties inherent in amplifying rare sequences. The critical step is sample partitioning. By separating each DNA template, 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.

There are currently two types of Digital PCR platforms:

  • Droplet Digital PCR (ddPCR), for example, Bio-Rad’s QX200™ Droplet Digital™ PCR System, utilizes microfluidics to emulsify samples in oil, creating reproducible droplets to be processed and analyzed by fluorescence.
  • 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.
Applications and Considerations of Digital PCR

Digital PCR was originally developed as a technique to investigate rare variants of minority targets such as mutations, while in the presence of large numbers of wild-type sequences. 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 preparation.

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. This study demonstrates that Droplet Digital PCR (ddPCR™) with Bio-Rad’s QX100 ddPCR system (this has now been upgraded to the QX200™ Droplet Digital™ PCR System) can be easily incorporated into the library preparation workflow to accurately quantify and balance sequencing libraries on Illumina sequencers.

Droplet-Digital

Figure 2: NGS sample quantification using Bio-Rad’s QX200™ Droplet Digital™ PCR System

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.

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, and screening for tumor and pathogen resistance.

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.

PCR Reagents & Considerations

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.

The water source is vital for successful PCR experiments. It must be nuclease-free, and 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, use ultrapure bottled water. See the SelectScience Laboratory Water Purification Buying Guide for system options and considerations.

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. You can purchase dNTPs as a premixed solution of dATP, dTTP, dCTP and dGTP, or they can be supplied individually. The quality of dNTPs is vital to the success of PCR experiments; once purchased, do not aliquot dNPTs 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.

BUYING GUIDE TIP: When choosing DNA polymerases, it is 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.

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. See the SelectScience DNA and RNA Purification and Analysis Buying Guide for help with choosing the best equipment and kits for your PCR template preparation.

DNA Polymerases

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.

BUYING GUIDE TIP: 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. 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 GoTaq® 2-Step RT-qPCR System, from Promega, provides a ready-to-use kit for analyzing a wide range of RNA targets by combining the high-activity of GoScript™ Reverse Transcription System with the ultra-bright fluorescence of GoTaq® qPCR Master Mix.

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.

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.

PCR Consumables & Considerations

Not all plastic-ware 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, such as in Sarstedt’s White Multiply® PCR Plates (see figure 3 below) 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.

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Figure 3: White Multiply® PCR Plates and highly transparent tapes for qPCR

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.

BUYING GUIDE TIP: When purchasing consumables, consider compatibility with your PCR instrumentation and the level of your throughput.

Plate sealing is commonly achieved using either adhesive, or heat sealing films and foils.

PCR Clean-up & Considerations

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. Many commercial kits are based on spin-column technology and can be used for either gel extraction, for example, QIAGEN’s MinElute Gel Extraction Kit (50) or direct purification of PCR products, for example, QIAGEN’s QIAquick 96 PCR Purification Kit (24).

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.

GC-Rich PCR

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. Several optimization kits incorporating reagents and a variety of buffers are currently marketed. For example, the Taq PCR Core Kit, by QIAGEN, has a Q-Solution for amplification of GC-rich templates.

DNA polymerases that have been engineered specifically for the amplification of GC-rich DNA are available. The unique properties of such enzymes may include enhanced processivity and improved tolerance to DNA melting agents.

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. See the DNA and RNA Purification and Analysis Buying Guide for further information on crude sample preparation kits.

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.

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.

Automated PCR

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, 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, 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.

BUYING GUIDE TIP: 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.

Multiplex PCR

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. This tech report focuses on the use of the thermal gradient feature on the CFX96™ real-time PCR detection system to optimize reverse transcription (RT) and fast PCR conditions for a four-target multiplex PCR assay.

BUYING GUIDE TIP: 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.

In clinical diagnostics, another area of potential is for non-invasive pre-natal screening for fetal chromosomal abnormalities, 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.

Summary

Visit the SelectScience PCR product and user product reviews directory for an overview of the latest products from leading manufacturers.

Keep up to date with the latest techniques and advances in PCR by visiting the SelectScience PCR pages for application notes, videos and the latest news.

Editor's Picks

Kerry Parker Kerry Parker
Editor-in-Chief

QX200™ Droplet Digital™ PCR System (Bio-Rad)

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4 out of 5

“The droplet technology has been pretty straightforward to use and teach to others. It has thus far replicated several differential gene expression and copy number studies in our lab.”
Robert Thompson, University of Michigan

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3Prime PCR Thermal Cyclers (Bibby Scientific)

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4 out of 5

“This is an easy to use and low-cost product. Economically, it’s an ideal choice for routine PCR works. The warranty is among the best in the market.”
Arnold Gaje, University of the Philippines Visayas

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Takara PCR Kits and Reagents (Takara Clontech)

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5 out of 5

“Products are pretty cheap and effective. Yield and quality is more than expected.”
Sateesh Maddirevula, Chungnam National University


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Mastercycler® Series (Eppendorf)

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4 out of 5

“Since the heating and cooling is very fast, each PCR reaction takes a short amount of time. It also can control several units at the same time. So it saves money.”
Chi Li Yu, The University of Iowa


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QIAquick 96 PCR Purification Kit (QIAGEN)

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5 out of 5

“It is a great product for purifying PCR products and getting rid of primers and dNTPs.”
Ivo Teneng, Lovelace Respiratory Research Institute




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GoTaq® 2-Step RT-qPCR System (Promega)

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4 out of 5

“The kind of product I recommend to any routine PCR laboratory; It made my work easy and I had good results using it.”
Daniel Filho, Universidade Federal do Acre


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