The polymerase chain reaction (PCR) is an in vitro molecular biology method used to amplify DNA sequences. Conceptualized in 1983 by Dr. Kary Mullis, who then went on to receive the Nobel Prize in Chemistry in 1993, PCR 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.
PCR has revolutionized scientific research, and yet the basic principle of PCR remains elegantly simple. It involves a three-step reaction of thermal denaturation, primer annealing and primer extension.
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.
There are numerous variations to a PCR, but the basic 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.
The PCR template quality and quantity often determine whether PCR amplification is successful. Because PCR consists of multiple cycles of enzymatic reactions, it is more sensitive to impurities such as proteins, phenol, chloroform, salts, ethanol, EDTA, and other chemical solvents than single-step, enzyme-catalyzed processes. There are numerous PCR sample preparation kits available to purify nucleic acids from a wide variety of sample materials, such as whole blood, bacterial cells, viruses and tissues. The ‘How to Buy DNA/RNA Purification and Quantification Technology’ eBook provides useful information on the best equipment and kits for your PCR template preparation.
Good primer design ensures correct amplification and, therefore, a 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.
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 with 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 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.
Several types of DNA polymerases 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.
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 dNTPs 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.
PCR master mixes provide the key ingredients necessary for performing PCR in a pre-mixed 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.
While the basic PCR procedure is still commonly used in many laboratories, PCR has evolved far beyond simple amplification and detection. Many variations of the original PCR method have been described, which include:
4.1 Hot Start PCR
In conventional PCR, nonspecific primer annealing can occur at low temperatures, and although the activity of the DNA polymerase is significantly compromised, extension can still occur. This results in undesired PCR products and lower yields of desired product. Hot start PCR involves blocking DNA polymerase activity at low temperatures and is used to reduce nonspecific amplification and improve the performance of PCR. Hot start PCR can be achieved by simply withholding the polymerase until higher temperatures are attained. Other methods include using physical barriers, such as wax beads, to segregate key reaction components, using specific inhibitors or antibodies to block DNA polymerase activity, and chemically modifying other components such as dNTPs and primers.
4.2 Reverse Transcriptase PCR
The DNA polymerases used for basic PCR require a DNA template. Numerous applications such as gene expression profiling, however, would benefit from amplification of RNA. The reverse transcriptase PCR (RT-PCR) was, thus, developed. In RT-PCR, the RNA sample must first be reverse transcribed to cDNA using a reverse transcriptase enzyme. The resulting cDNA can then be used for subsequent PCR amplification.
4.3 Quantitative PCR (qPCR)
Quantitative PCR (qPCR) enables accurate quantification of starting amounts of DNA, cDNA, and RNA targets. Unlike the conventional PCR where quantification occurs as a separate step after the amplification of DNA, in qPCR, a fluorescent reporter dye enables a real-time detection of PCR products during each amplification cycle. An increase in nucleic acid amplification is proportional to the increase in fluorescence signal. Because qPCR detects the amount of nucleic acids as the reaction progresses, it provides a wide, linear, dynamic range; demonstrates high sensitivity; and is very quantitative as its name suggests. Frequently, qPCR is combined with reverse transcriptase PCR to quantify messenger RNA, microRNA and non-coding RNA.
4.3.1 qPCR Fluorophores
Reporters in qPCR are either (1) DNA-binding dyes or (2) fluorescently-labeled probes.
Double-stranded DNA (dsDNA)-binding dyes bind reversibly, but tightly to dsDNA by intercalation and/or minor groove binding. Before amplification, during the presence of single-stranded DNA, these dyes emit very low intensity signals that are undetectable. However, at the end of the elongation step, as dsDNA levels increase, the dyes bind to dsDNA and fluoresce high-intensity signals.
Among the commercially available fluorescent dyes, the SYBR® Green is by far the most widely used dsDNA-specific dye for real-time PCR. SYBR Green binds all dsDNA molecules, emitting a fluorescent signal of a defined wavelength on binding.
DNA binding dyes cannot be used for multiplex reactions because fluorescent signals from different amplicons cannot be distinguished from one another. Parallel reactions can, however, be set up to examine multiple genes in a qPCR assay with SYBR Green.
Using dsDNA-binding dyes will provide the simplest and cheapest option for real-time PCR, but the principal drawback is that both specific and non-specific products generate signal. High PCR specificity is required when using fluorescent dyes, and a post-PCR dissociation (melting) curve analysis should be carried out to confirm that the fluorescence signal is generated only from target templates and not from the formation of non-specific PCR products.
This useful application note by Analytik Jena offers a qPCR performance comparison of six different ready-to-use master mixes with an intercalating dsDNA-binding dye for the qPCR of human GAPDH. It is interesting to note the differences in performance in the very same experiments by merely changing the reagents.
2. Fluorescently-labeled probes
Fluorescently-labeled probes bind to a specific region of the template DNA and provide a highly sensitive and specific method of detection. Several probes based on different chemistries are available for real-time detection; these include:
I. TaqMan® probes:
TaqMan hydrolysis probes employ the 5’ → 3’ exonuclease activity of Taq. Carrying a fluorophore and a quencher, they bind to a specific oligonucleotide sequence of the template DNA. As the probe binds to a pre-determined region of the DNA template; the fluorophore is attached at the 5' end of the probe and the quencher dye is located at the 3' end. When intact during annealing, the fluorophore’s signal is quenched by the close proximity of the quencher. During amplification, the dsDNA-specific 5' → 3' exonuclease activity of Taq cleaves off the reporter. Now separated from the quencher, the reporter fluoresces, its signal proportional to the amplified product.
II. Molecular beacons:
Molecular beacons are dual-labeled probes with a fluorophore attached at the 5' end and a quencher dye attached at the 3' end. The probes are designed such that the ends have complementary sequencing. Therefore, when in solution, the two ends of the probe hybridize and form a hairpin structure. With the fluorophore and quencher in close enough proximity, the fluorescent signal is quenched. When the probe binds to the target sequence, the stem opens and the fluorophore and quencher separate. This generates a fluorescent signal in the annealing step that is proportional to the amount of PCR product. Molecular beacons add another level of specificity to real-time PCRs and are particularly useful for allelic discrimination experiments.
III. Dual hybridization probes:
These involve using two labeled oligonucleotide probes that are designed to bind to two adjacent regions on target DNA. The first probe carries a donor dye at its 3' end, while the second carries an acceptor dye at its 5' end. The donor and the acceptor dyes are selected such that they exhibit FRET (fluorescence resonance emission transfer). During annealing, excitation is performed at a wavelength specific to the donor dye, and the reaction is monitored at the emission wavelength of the acceptor dye. Fluorescence detected during the annealing phase of PCR is proportional to the amount of amplification produced.
4.3.2 Real-time thermal cyclers
A qPCR experiment requires a specialized real-time PCR thermal cycler equipped with fluorescence detection modules used to monitor the fluorescence as amplification occurs. Listed below are considerations for purchasing a real-time PCR thermal cycler:
The key feature of real-time PCR is precise fluorescence measurements, making optics a very important consideration when purchasing a qPCR thermal cycler. It is important that your thermocycler transmits homogeneous excitation and illumination across all your samples in, say, a 96-well experiment, for reproducibility and consistency. The qTOWER3 by Analytik Jena, for example, includes a patented fiber-optic shuttle system with a unique light source comprising of four different-colored (RGBW) LEDs, thus enabling excitation of all known fluorescent dyes in your experimental plan.
To save time and to ensure constant experimental conditions, it has become crucial to consider multiplexing PCRs. Find out how many different fluorescent-labeled probes can be used in your qPCR thermocycler. For instance, qTOWER3 offers multiplexing capabilities with six different probes ranging from blue to near-infrared wavelengths.
In addition to fluorescence detection in a qPCR experiment, the heat-enabled amplification itself determines the success of the experiment. Enquire about your desired instrument’s temperature control, i.e. if set at 65⁰C, how precisely is 65⁰C maintained across all wells? Plus, the ramping rate of the instrument – the time it takes for the instrument to change temperature settings – becomes a factor in completing the protocol faster. Analytik Jena’s qTOWER3, for example, exhibits a temperature control precision of ± 0.1⁰C and a ramping rate of 8⁰C/s.
With extreme temperature changes across an experiment, condensation can become an issue, often resulting in a loss of samples. Check if the qPCR device of your choice is equipped with a motorized, heated lid that curbs condensation.
Your experiment is only as good as the light detected from the fluorophores. Enquire about the detectors in the device of your choice, the read-out times and the compatibility of different color modules, i.e. the use of DNA-binding dyes, molecular beacons or dual hybridization probes with your experiments. A high-sensitivity photomultiplier tube in qTOWER3 detects signals with a read-out time of six seconds for 96 wells, irrespective of the number of dyes. With 12 color-, FRET- and protein-based modules in the qTOWER3, you can plan experiments with a diverse set of reporters. Plus, the detection module can accept up to six different color filter modules, enabling retrofitting for future developments.
If your instrument is compatible with a 384-well temperature block, it instantly adds high-throughput capabilities to your experiments, miniaturizing individual reactions. The qTOWER3 84 is adapted to the 384-well format, with up to six-fold multiplexing capabilities, and contains 16 scanning fibers, enabling extremely fast read-outs in six seconds.
Many modern real-time thermal cyclers now possess an inbuilt software analysis system, coupled with a touch screen or computer integration. To instantly obtain results, consider a software package that calculates Ct values and provides graphs you can directly use. The easy-to-use qPCRsoft package in the qTOWER3 covers an entire spectrum of analyses and doesn’t require any additional licenses to use.
The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describes the minimum information necessary for evaluating real-time PCR experiments and helps promote better experimental practice. A practical guide to publishing data that conform to the MIQE guidelines can be read here. Enquire with the manufacturer if you can obtain a copy of the MIQE-compliant documentation of the instrument you are purchasing.
4.3.3 Applications of qPCR
Real-time PCR applications include gene expression analysis, validation of DNA microarrays, single nucleotide polymorphism (SNP) genotyping, copy number variation analysis, allelic discrimination analysis, drug metabolizing enzyme (DME) analysis, clinical molecular diagnostics, viral load quantification, and pathogen detection.
When analyzing gene expression using qPCR, some genes are expressed at very high copy numbers, while some others may be expressed at very low copy numbers. With lower copy numbers, obtaining equal-volume aliquots containing a copy of the gene becomes a challenge, whereas with very high copy numbers, the reagents don’t suffice or there’s too much background interference. This technical note by Analytik Jena details an experiment on the human GAPDH gene and demonstrates a wide linear range with accurate quantification across 10 orders of magnitude.
In the food industry, significant efforts are being made to detect adulteration or non-declared constitutes of animal origin in food in order to comply with regulatory standards and, in some countries, religious and health laws. Real-time PCR forms a convenient method to identify traces of animal DNA in food, for example, to determine the origin of gelatin in gummy bears or to identify traces of pork in rice. This useful downloadable application note outlines the types of qPCR experiments performed to test for sources of food products from different animals such as sheep, goat, cow and so on. The method also goes on to describe detection of food-borne pathogens, such as salmonella, using qPCR.
4.4 Digital PCR
Digital PCR (dPCR) is a technology that provides absolute counts of target DNA, as well as increased sensitivity, precision and reproducibility, compared to qPCR. dPCR has the potential to have a major impact on molecular analyses, ranging from clinical applications, such as biomarker analysis, viral detection, prognostic monitoring and fetal screening, to research applications such as phage-host interactions and intracellular profiling. It can also be applied to assist with the library preparation needed for next-generation sequencing.
4.4.1 Digital PCR Concept
The basic methodology with dPCR is to partition a DNA sample into thousands or tens of thousands of separate reaction chambers so that each contains one or no copies of the sequence of interest. Similar to qPCR, the amplicons are then hybridized with fluorescence probes, which allows the detection of sequence-specific products. PCR is performed in each partition, scoring a positive or negative for the presence of the target sequence based on its fluorescence, and returning an absolute value of DNA concentration.
dPCR improves upon the sensitivity of qPCR and enables the detection of signals by overcoming the difficulties inherent in amplifying rare sequences. The critical step is sample partitioning. By separating each DNA template, this technique, essentially, performs individual amplification reactions identified with fluorescence probes. Unlike qPCR, dPCR does not rely on the number of amplification cycles to determine the initial amount of template DNA in each sample. Rather, it relies on Poisson statistical analysis to determine absolute quantity of template DNA.
4.4.2 Digital PCR Platforms
There are currently two types of digital PCR platforms:
In this SelectScience video interview, Carolyn Reifsnyder, Bio-Rad Laboratories, Inc., introduces new assays for genome edit detection using the droplet digital PCR system, as well as the new ddSEQ™ Single-Cell Isolator, at the AACR conference in 2017.
4.4.3 Applications of Digital PCR
Digital PCR was originally developed as a technique to investigate rare variants of minority targets such as mutations, while in the presence of large numbers of wild-type sequences. The enhanced sensitivity and dynamic range offered by dPCR have facilitated the use of this method in a multitude of applications. Some examples of applications include copy number variation, rare sequence detection, gene expression and miRNA analysis, single-cell analysis, pathogen detection and next-generation sequencing sample preparation.
In an exclusive SelectScience editorial article, Dr. Bruce Conklin, Gladstone Investigator, and Professor in the Division of Genomic Medicine at University of California, San Francisco, shares his research on rare mutation detection. The Conklin lab has developed a robust, sensitive protocol to detect single nucleotide substitutions using digital PCR.
Next-Generation Sequencing (NGS): Studies have found that dPCR quantification is a more accurate and precise method for quality control of NGS libraries than conventional qPCR methods. NGS library quality control is essential for optimizing sequencing data yield, thereby increasing efficiency and throughput while lowering cost. Predominantly, NGS libraries are quantified using qPCR and their size determined by gel or capillary electrophoresis. However, these techniques have limitations and the steps to rectify them can be time-consuming and expensive. By adopting dPCR technology, it is possible to avoid the high cost of failed runs and suboptimal data yields.
Clinical diagnostics: Traditional PCR used extensively for clinical diagnostic applications suffers from lack of sensitivity. Digital PCR, however, can detect minute changes in gene sequence or quantity; it also provides a definitive, quantitative measurement of nucleic acid. This has potential for many areas of diagnostics, including non-invasive tumor mutation detection from body fluid analysis, and screening for tumor and pathogen resistance.
In this SelectScience video, Emil Christensen from Aarhus University, Denmark, discusses the use of droplet digital PCR (ddPCR) in monitoring the progression of bladder cancer in patients.
Figure 5. Emil Christensen anlyzes liquid biopsies in urine and plasma samples to detect mutations in bladder cancer. Watch the video here.
4.5 Other Variations of PCR
Variations to PCR can be achieved by making small alterations to the PCR protocol and/or the components of the PCR. Some of the most common types include:
Long PCR: In basic PCR, the efficiency of amplification, and therefore the yield of desired amplified products, decreases significantly as the amplicon size increases over 5kb. Amplification of DNA fragments larger than 5kb is desirable for numerous applications, including genome cloning, sequence mapping and gene cluster studies. Long PCR uses a blend of thermostable DNA polymerases and allows for the amplification of longer fragments.
Multiplex PCR: Multiplex PCR involves using multiple sets of primers for the amplification of several targets in a single PCR experiment. It is commonly used for pathogen identification, high-throughput SNP genotyping, mutation and gene deletion analysis, and also in forensic applications for human identification studies.
Colony PCR: Colony PCR is commonly used following transformation to screen colonies for the desired plasmid. In colony PCR, following the addition of a small quantity of cells to the PCR mix, the temperature and length of the initialization and/or denaturation steps are adapted to release the DNA from the cells, which is then available for amplification.
Nested PCR: In nested PCR, a second round of amplification is performed following conventional PCR, using a second set of primers that are specific to a sequence found within the DNA of the initial conventional PCR amplicon. The use of a second amplification step with ‘nested’ primers results in a reduction of nonspecific binding and increases the amount of amplicon produced.
Touchdown PCR: Touchdown PCR (TD-PCR) involves using a cycling program with varying annealing temperatures. In TC-PCR, an annealing temperature that is above the melting temperature of the primer is used for the initial cycle. It is then decreased in increments for subsequent cycles. The primer will anneal at the highest temperature that it is able to tolerate and is least permissive of nonspecific binding. TD-PCR therefore enhances the specificity of the initial primer-template duplex formation and hence the specificity of the final PCR product.
Allele-Specific PCR: Allele-Specific PCR (AS-PCR) is a convenient and reliable method for genotyping SNPs and mutations. In conventional PCR, primers are chosen from an invariant part of the genome and might be used to amplify a polymorphic area between them. In AS-PCR, at least one of the primers is designed from a polymorphic area with the mutation(s) located near its 3’-end. Under stringent conditions, the mismatched primer will not initiate replication, whereas a matched primer will permit amplification. The appearance of an amplification product therefore indicates the genotype.
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.
PCR tubes, purchased individually or in strips, are ideal for low-throughput PCR, whereas PCR plates are ideal for high-throughput. PCR plates often need to be sealed to prevent evaporation during PCR, reduce cross-contamination and/or enable efficient transport and storage. Plate sealing is commonly achieved using either adhesive or heat-sealing films and foils.
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 quickest, most convenient and reproducible approach to clean PCR products. You can find peer-reviewed kits in SelectScience’s product directory. 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 or direct purification of PCR products. More information on the best DNA/RNA kit for your downstream application is available in our ‘How to Buy DNA/RNA Purification and Quantification Technology’ eBook.
PCR with GC-rich (>60%) templates 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.
To yield a successful GC-rich PCR, specific reagents are added to the reaction mix. 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, however, 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. The human blood possesses several major inhibitors of PCR, namely, 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. Pre-treatment of samples and using specialized DNA extraction kits are ways to overcome the complexities of using crude samples for PCR.
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 discovery engineering of thermostable polymerases and the development of automated thermocyclers, high-throughput real-time PCR instruments and digital PCR methods are among the most notable PCR advances of recent years.
As projects get larger, consistency, speed and performance become increasingly important. Reliable tools for automated setup of reactions and sensitive platforms for fragment detection are making their way into laboratories. Also, researchers now seek new tools which enable higher-throughput PCR at an increased speed, with smaller reaction volumes and lower costs.
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.
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.
The SelectScience product directory provides you with a convenient list of PCR-related products, kits and reagents. Plus, you can read reviews on the products to learn about the opinions of your peers.