A comprehensive introduction to PCR and qPCR methods, including video instructions and example protocols.
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Introduction to PCR
Polymerase chain reaction (PCR) is a relatively simple technique that amplifies a DNA template to produce specific DNA fragments in vitro. Traditional methods of cloning a DNA sequence into a vector and replicating it in a living cell often require days or weeks of work, but PCR amplification of DNA sequences takes only a few hours. While most biochemical analyses, including the detection of radioisotope nucleic acids, require the input of significant amounts of biological material, the PCR process requires very little. Therefore, PCR can achieve more sensitive detection and higher amplification levels of specific sequences in a shorter time than previously used methods. These features make this technique extremely useful not only in basic research, but also in commercial applications, including genetic identity testing, forensics, industrial quality control, and in vitro diagnostics. Basic PCR is common in many molecular biology labs, where it is used to amplify DNA fragments and detect DNA or RNA sequences in a cell or the environment. However, PCR has evolved far beyond simple amplification and detection, and many extensions of the original PCR method have been described. This chapter provides an overview of the different types of PCR methods, applications and optimization.
The PCR process was originally developed to amplify short segments of a longer DNA molecule (Saiki et al. 1985). A typical amplification reaction includes target DNA, thermostable DNA polymerase, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, and magnesium. Once assembled, the reaction is placed in a thermal cycler, an instrument that subjects the reaction to a range of different temperatures for specific periods of time. This series of temperature and time adjustments is known as the gain cycle. In theory, each PCR cycle doubles the amount of target sequence (amplicon) in the reaction. In theory, ten cycles multiply the amplicon by a factor of about a thousand; 20 cycles, with a factor of more than a million in a few hours.
Each PCR cycle includes template denaturation, primer annealing, and primer extension steps. The first step denatures the target DNA by heating it at 94°C or more for 15 seconds to 2 minutes. The denaturation process separates the two intertwined strands of DNA, creating a single-stranded DNA template required for replication by thermostable DNA polymerase. In the next step of the cycle, the temperature is lowered to approximately 40–60°C. At this temperature, oligonucleotide primers can form stable associations (hybridize) with denatured target DNA and serve as primers for DNA polymerase. This step takes about 15 to 60 seconds. Finally, the synthesis of new DNA begins when the reaction temperature rises to the optimum level for DNA polymerase. For most thermostable DNA polymerases, this temperature is in the range of 70 to 74 °C. The extension step takes about 1 to 2 minutes. The next cycle begins with a return to 94°C for denaturation.
Each cycle step must be optimized for each template-primer pair combination. If the temperatures during the annealing and extension steps are similar, these two steps can be combined into one step where both annealing and primer extension take place. After 20 to 40 cycles, the amplified product can be analyzed for size, amount, sequence, etc. or used in further experimental procedures.
Thermostable DNA polymerases used for simple PCR require a DNA template and as such the technique is limited to the analysis of DNA samples. However, there are numerous cases where RNA amplification would be desirable. To apply PCR to the study of RNA, the RNA sample must first be converted to cDNA to provide the required DNA template for the thermostable polymerase (Figure 1). This process is called reverse transcription (RT), hence the name RT-PCR.
Reverse transcriptases (RTs) are RNA-directed DNA polymerases that were first identified as part of the life cycle of retroviruses (Temin and Mizutani, 1970, Baltimore, 1970). RTs catalyze the synthesis of a single copy DNA (cDNA) of a target RNA molecule using a reverse transcription primer, dNTPs and Mg2+mangan2+as a cofactor. Reverse transcriptases have been adapted for use in a variety of in vitro applications, including real-time and endpoint RT-PCR, the generation of labeled cDNA probes, and the construction of cDNA libraries. The ideal reverse transcriptase is robust (highly active under a variety of conditions) and converts all of the RNA prepared in the sample to cDNA, regardless of its amount, length, or secondary structure.
The most characteristic RTs used in molecular biology are retroviral RTs: avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (M-MLV or MuLV). Genetic engineering and the development of proprietary buffers to improve RT have led to the commercial availability of novel enzymes that offer superior performance to these natural RTs.
AMV and M-MLV reverse transcriptases are generally used to generate a DNA copy from an RNA template using random primers, oligo(dT) primers, or sequence-specific primers. Some thermostable DNA polymerases (for example, Tth DNA polymerase) possess reverse transcriptase activity, which can be activated by using manganese instead of magnesium as a cofactor (Myers and Gelfand, 1991). After this initial step of reverse transcription to produce the cDNA template, basic PCR is performed to amplify the target sequence.
The quality and purity of the RNA sample are critical to the success of RT-PCR. Total RNA or poly(A)+ RNA can be used as starting template, but both must be intact and free of contaminating genomic DNA. Specific capture of poly(A)+ RNA will enrich the target message so that fewer reverse transcription reactions are required for subsequent amplification. The efficiency of the first strand synthesis reaction, which may be related to the quality of the RNA, will also significantly affect the amplification results.
GoScript™ Reverse Transcriptase is an optimized formulation of M-MLV reverse transcriptase and buffers designed for efficient and reproducible first-strand cDNA synthesis from a full range of rare and abundant transcripts, even with difficult templates and in the presence of inhibitors. PCR. GoScript is qualified for use in qPCR and is compatible with GoTaq® RT-qPCR systems. GoScript is available in convenient mixes with Oligo(dT) primers or random primers, as part of a complete kit, and as a standalone enzyme.
RT PCR products and resources
GoScript® Reverse Transcriptase is available as a stand-alone reverse transcriptase or as one- and two-step RT-PCR kits.
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PCR with hot start
Hot-start PCR is a commonly used technique to reduce non-specific amplification due to the assembly of amplification reactions at room temperature. At room temperature, PCR primers can hybridize to template sequences that are not perfectly complementary. Because thermostable DNA polymerases are active at these low temperatures (although the activity is less than 25% in most cases), the polymerase can extend mishandled primers. This newly synthesized region then acts as a template for primer extension and synthesis of unwanted amplification products. However, if the reaction is heated to temperatures >60°C before starting the polymerization, the stringency of the primer annealing is increased and the synthesis of unwanted PCR products is prevented or reduced.
Hot start PCR can also reduce the amount of primer dimer synthesized by increasing the stringency of primer annealing. At lower temperatures, PCR primers can attach to each other via regions of complementarity, and DNA polymerase can extend the annealed primers to produce a primer dimer, which often appears as a diffuse band of about 50 to 100 bp in an ethidium bromide-stained gel. . The formation of non-specific and primer-dimer products can compete for the availability of reagents with amplification of the desired product. Therefore, hot-start PCR can improve the efficiency of specific PCR products.
To perform manual warm PCR, the reactions are pooled on ice or at room temperature, but the critical component is omitted until the reaction warms to 60-65 °C, at which point the missing reagent is added. This omission prevents the polymerase from extending primers until a critical component is added at the highest temperature where primer annealing is most stringent. However, this method is cumbersome and increases the risk of contamination. Another approach, which is less labor intensive, involves reversible inactivation or physical separation of one or more critical components in the reaction. For example, magnesium or DNA polymerase can be sequestered in a wax bead, which melts when the reaction is heated to 94°C during the denaturation step, so that the component is only released at higher temperatures (Carotherset al. 1989; Krishnaet al. 1991; Clark, 1988). DNA polymerase can also be kept in an inactive state by binding to an oligonucleotide, also known as an aptamer (Lin and Jayasena, 1997; Dang and Jayasena, 1996) or an antibody (Scaliceet al. 1994.; sea beltet al. 1994). This bond is broken at higher temperatures, releasing functional DNA polymerase. Finally, DNA polymerase can be kept in an inactive state by chemical modification (Moretti, T.et al1998).
Hot Start PCR Products and Resources
GoTaq® G2 Hot Start Taq is available as a stand-alone enzyme or as a master mix. In this formulation, Taq polymerase is bound to proprietary antibodies that block its activity. Activity is restored during initial denaturation, allowing a hot start of PCR.
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Long range PCR
Amplification of long DNA fragments is desirable for a number of applications, such as physical mapping applications (Rose, 1991) and direct genome cloning. Although basic PCR works well at amplifying smaller fragments, the amplification efficiency (and thus the yield of amplified fragments) decreases significantly as the size of the amplicon exceeds 5 kb. This decrease in efficiency can be attributed to the accumulation of truncated products, which are not suitable substrates for additional cycles of amplification. These products appear as smeared bands, rather than discrete ones, on the gel.
In 1994, Wayne Barnes (Barnes, 1994) and other researchers (Chenget al. 1994) investigated the factors influencing polymerization in larger regions of DNA using thermostable DNA polymerases and identified key variables influencing the performance of longer PCR fragments. They devised an approach that uses a mixture of two thermostable polymerases to synthesize longer PCR products. The first polymerase lacks 3' → 5' exonuclease activity (correction); the second enzyme, which is present in low concentration, has a strong corrective effect. Presumably, when a non-proofreading DNA polymerase (e.g., Taq DNA polymerase) incorrectly incorporates a dNTP, further elongation of the newly synthesized DNA will proceed very slowly or stop altogether. A proofreading polymerase (e.g., Pfu DNA polymerase or Tli DNA polymerase) serves to remove the misincorporated nucleotide, allowing DNA polymerases to proceed with new strand elongation.
While the use of two thermostable DNA polymerases can significantly increase the yield, other conditions can significantly affect the yield of longer PCR products (Chenget al. nineteen ninety five). Of course, longer extension times can increase the yield of longer PCR products because fewer partial products are synthesized. The extension times depend on the length of the goal; Times of 10 to 20 minutes are common. The quality of the template is also crucial. Purification of the template, which is favored at higher temperatures and lower pH, will result in more partial products and a lower overall yield. In long PCR, denaturation time was reduced to 2 to 10 seconds to reduce template clearance. Additives, such as glycerol and dimethyl sulfoxide (DMSO), also help reduce wire stripping and primer annealing temperature, mitigating some of the effects of high-temperature scrubbing. Chenget al. He also found that lowering the potassium concentration by 10-40% increased the amplification efficiency of longer products (Chenget al. 1995).
GoTaq® Long PCR Master Mix contains hotstart Taq in a specially formulated mix with a proprietary heat-stable proofreading polymerase. This optimized mixture of enzymes allows efficient amplification of up to 40 kb of lambda DNA or 30 kb of human genomic DNA.
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qPCR en RT-qPCR
Quantitative endpoint PCR
PCR and RT-PCR are generally used in a qualitative format to evaluate biological samples. However, a wide range of applications, such as determining viral load, measuring response to therapeutic agents and characterizing gene expression, would be improved by quantitative targeting. In theory, this should be easy to achieve, given the exponential nature of PCR, because there is a linear relationship between the number of amplification cycles and the logarithm of the number of molecules. In practice, however, amplification efficiency decreases due to contaminants (inhibitors), competitive reactions, substrate depletion, polymerase inactivation and target annealing. As the number of cycles increases, the amplification efficiency decreases, eventually resulting in a plateau effect.
Quantitative PCR usually requires measurements before the plateau phase so that the relationship between cycle number and molecule is relatively linear. This point needs to be determined empirically for different reactions because of the numerous factors that can affect amplification efficiency. Since the measurement is performed before the reaction plateaus, quantitative PCR uses fewer amplification cycles than regular PCR. This can cause problems in discovering the final product as there are fewer products to discover.
To check amplification efficiency, many applications are designed to include an internal standard in the PCR. One such approach includes a second primer pair specific for a "housekeeping" gene (i.e., a gene that has constant expression levels between compared samples) in the reaction (Gaudette and Crain, 1991; Murphyet al.1990). Housekeeping gene amplification verifies that the target nucleic acid and reactants were of acceptable quality, but does not account for differences in amplification efficiency due to differences in product size or sample annealing efficiency between the internal standard and the target being quantified.
The concept of competitive PCR, a variation of quantitative PCR, is a response to this limitation. In a competitive PCR reaction, a known amount of control sample is added. This template is amplified using the same primer pair as the experimental target molecule, but produces a recognizable product (e.g., different size, restriction digestion pattern, etc.). The amounts of control and test product are compared after amplification. While these approaches control target nucleic acid quality, buffer components, and primer annealing efficiency, they have their limitations (Siebert and Larrick, 1993; McCullochet al.1995), including the fact that many rely on final analysis by electrophoresis.
There are numerous solid phase and fluorescence assays for measuring the amount of amplification product formed in each reaction, but these often fail to distinguish the amplified DNA of interest from non-specific amplification products. Some of these tests are based on blotting techniques, which introduce another variable due to nucleic acid transfer efficiency, while other tests have been developed to eliminate the need for gel electrophoresis while providing the necessary specificity. Real-time PCR, which provides the ability to view the results of each amplification cycle, is a popular way to overcome the need for electrophoresis analysis.
Quantitative real-time PCR
The use of fluorescently labeled oligonucleotide primers or probes or fluorescent DNA binding dyes to detect and quantify PCR products allows the performance of real-time quantitative PCR. Custom-designed instruments perform thermal cycling to enhance target and fluorescence detection and monitor the accumulation of PCR products. DNA-binding dyes are easy to use, but do not differentiate between specific and non-specific PCR products and do not lead to multiplex reactions. Fluorescently labeled nucleic acid probes have the advantage of reacting only with specific PCR products, but can be expensive and difficult to design. Some qPCR technologies use fluorescently labeled PCR primers instead of probes.
Using fluorescent dyes that bind DNA is one of the simplest qPCR approaches. Dye is simply added to the reaction and fluorescence is measured in each PCR cycle. Since the fluorescence of these dyes increases dramatically in the presence of double-stranded DNA, DNA synthesis can be monitored as an increase in fluorescence signal. However, often some preparatory work needs to be done to ensure that the PCR conditions produce only the specific product. In the following reactions, specific amplification can be confirmed by melting curve analysis. Thermal melting curves are generated by allowing the entire product to form double-stranded DNA at a lower temperature (approximately 60°C) and slowly increasing the temperature to denaturation levels (approximately 95°C). The order and length of the product influence the melting temperature (Tm), so the melting curve is used to characterize the homogeneity of the amplicon. Non-specific amplification can be recognized by broad peaks in the melting curve or peaks with unexpected Tm values. By distinguishing between specific and non-specific amplification products, the melting curve adds a quality control aspect during routine use. Melting curve generation is not possible with assays that rely on the 5′ → 3′ exonuclease activity of Taq DNA polymerase, such as the probe-based TaqMan® technology.
Some qPCR strategies use complementary nucleic acid probes to quantify target DNA. These probes can also be used to detect single nucleotide polymorphisms (Leeet al. 1993; Bernardoet al. 1998). There are several general categories of real-time PCR probes, including hydrolysis, hairpin, and single hybridization probes. These probes contain a complementary sequence that allows the probe to hybridize to the accumulated PCR product, but the probes may differ in the number and position of fluorescent reporters.
The hydrolysis probes are labeled with fluorine at the 5' end and a quencher at the 3' end, and since the two reporters are in close proximity, the fluorescence signal is quenched. During the hybridization step, the probe hybridizes to the PCR product generated in previous amplification cycles. The resulting probe:target hybrid is a substrate for the 5′ → 3′ exonuclease activity of DNA polymerase, which degrades the hybridized probe and releases fluoride.et al. 1991). Fluorine is released by the effects of the energy-absorbing quencher, and the progress of the reaction and the accumulation of PCR products are monitored by the resulting increase in fluorescence. With this approach, preliminary experiments should be performed before quantification experiments to demonstrate that the generated signal is proportional to the amount of the desired PCR product and that no non-specific amplification occurs.
Hairpin probes, also known as molecular beacons, contain inverted repeats separated by a sequence complementary to the target DNA. The repeats hybridize to form a hairpin structure, with the fluorine at the 5' end and the quencher at the 3' end in close proximity, resulting in a small fluorescence signal. The hairpin is designed so that the probe preferentially binds to the target DNA rather than retaining the hairpin structure. As the reaction progresses, increasing amounts of probe hybridize to the pooled PCR product, and as a result, the fluoride and quencher physically separate. The fluorine no longer extinguishes and the fluorescence level increases. One of the advantages of this technique is that hairpin probes are less likely to mismatch than hydrolysis probes (Tyagiet al. 1998). However, preliminary experiments must be performed to demonstrate that the signal is specific to the desired PCR product and that no non-specific amplification occurs.
Using simple hybridization probes involves two labeled probes, or alternatively one labeled probe and one labeled PCR primer. In the first approximation, the energy emitted by a fluorine from one probe is absorbed by a fluorine from another probe, which hybridizes nearby. In another approach, the emitted energy is absorbed by another fluorine incorporated into the PCR product as part of the primer. Both approaches result in increased fluorescence of the energy acceptor and decreased fluorescence of the energy donor. The use of hybridization probes can be further simplified by requiring only one labeled probe. This approach uses the quenching of fluorine by deoxyguanosine to produce a change in fluorescence (Crockett and Wittwer, 2001; Kurataet al. 2001). The labeled probe hybridizes so that the fluorine is in close proximity to G residues within the target sequence, and as probe hybridization increases, fluorescence decreases due to deoxyguanosine quenching. With this approach, probe placement is limited because the probe must be hybridized so that the fluorescent dye is close to residue G. The advantage of single hybridization probes is their ability to be multiplexed more easily than single hybridization probes. hydrolysis and hairpin using fluorine of different colors and probes with different melting temperatures (reviewed in Wittweret al. 2001).
Some qPCR strategies use complementary nucleic acid probes to quantify target DNA. These probes can also be used to detect single nucleotide polymorphisms (Leeet al. 1993; Bernardoet al. 1998). There are several general categories of real-time PCR probes, including hydrolysis, hairpin, and single hybridization probes. These probes contain a complementary sequence that allows the probe to hybridize to the accumulated PCR product, but the probes may differ in the number and position of fluorescent reporters.
qPCR products and resources
GoTaq® qPCR and RT-qPCR kits are available for dye or probe based real-time PCR methods. GoTaq qPCR systems contain BRYT Green Dye, which offers the highest amplification efficiency and higher fluorescence than SYBR Green.
GoTaq® Probe Systems are ready-to-use master mixes that simplify reaction assembly for hydrolysis probe-based detection.
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General Considerations for PCR Optimization
This discussion focuses on the use of Taq DNA polymerase in PCR as it is the most commonly used enzyme in PCR. Many of these suggestions also apply when using other DNA polymerases.
Magnesium is an essential cofactor for thermostable DNA polymerases, and magnesium concentration is a key factor that can influence the success of amplification. The concentration of the DNA sample, the chelating agents (e.g. EDTA or citrate) present in the sample, the dNTP concentration and the presence of proteins can influence the amount of free magnesium in the reaction. In the absence of sufficient free magnesium, Taq DNA polymerase is inactive. Excess free magnesium reduces enzyme fidelity (Eckert and Kunkel, 1990) and may increase the level of non-specific amplification (Williams, 1989; Ellsworthet al. 1993). For these reasons, researchers must empirically determine the optimal magnesium concentration for each target. To do this, you need to set up a series of reactions containing between 1.0 and 4.0 mM Mg.2+in steps of 0.5 to 1 mM and review the results to determine which concentration of magnesium produced the highest yield of product and the lowest amount of non-specific product. The effect of magnesium concentration and the optimal concentration range may vary depending on the individual DNA polymerase. For example, the effect of Pfu DNA polymerase seems to be less dependent on magnesium concentration, but when optimization is required, the optimal concentration is usually in the range of 2-6 mM.
Many DNA polymerases come with magnesium-free reaction buffer and a tube of 25 mM MgCl.2so you can adjust the Mg2+concentration to the optimum level for each reaction. Before preparing the reactions, make sure that the pre-use magnesium solution is completely dissolved and that you shake the magnesium solution for a few seconds before pipetting. Magnesium chloride solutions may form concentration gradients due to multiple freeze-thaw cycles, and vortexing is required to obtain a uniform solution. These two steps, while seemingly simple, eliminate the cause of many failed experiments.
Some scientists prefer to use reaction buffers that already contain MgCl.2at a final concentration of 1.5 mM. However, it should be noted that Huet al. (1992) reported variability in the performance of reaction buffers containing magnesium. The 0.6 mM changes in free magnesium observed in their experiments dramatically affected amplification yields in an allele-specific manner. The authors found that heating the buffer to 90°C for 10 minutes restored the homogeneity of the solution. They hypothesized that the magnesium chloride precipitated as a result of multiple freeze-thaw cycles.
Most reaction buffers consist of a buffering agent, usually a Tris-based buffer, and a salt, usually KCl. The buffer regulates the pH of the reaction, which affects the activity and fidelity of the DNA polymerase. Modest concentrations of KCl will increase DNA polymerase activity by 50-60% compared to activity in the absence of KCl; 50 mM KCl is considered optimal (Gelfand, 1989).
GoTaq® DNA Polymerase contains natural Taq DNA Polymerase in a proprietary formulation. Supplied with 5x green GoTaq® reaction buffer and 5x clear GoTaq® reaction buffer. 5X Green GoTaq® Reaction Buffer contains blue and yellow dyes that are separated during electrophoresis to monitor migration progress. The buffer also contains a compound that increases the density of the sample so that it sinks into the well of the agarose gel, allowing reactions to be loaded directly onto the agarose gel without the need to add dye. The blue dye migrates at the same rate as a 3-5 kb DNA fragment on a 1% agarose gel. The yellow dye migrates faster than primers (<50 bp) on a 1% agarose gel. GoTaq® Colorless 5X Reaction Buffer and GoTaq® Green 5X Reaction Buffer have the same formulation, except for the colors. GoTaq® Colorless 5X Reaction Buffer is recommended for any application where PCR amplimer absorbance or fluorescence measurements are performed without pre-cleaning. Both buffers are supplied at pH 8.5 and contain MgCl.2at a concentration of 7.5 mM for a final concentration of 1.5 mM.
GoTaq® Flexi DNA Polymerase is supplied with 5X Green GoTaq® Flexi Reaction Buffer and 5X Clear GoTaq® Flexi Reaction Buffer. The composition is identical to GoTaq® Green 5X Reaction Buffer and GoTaq® Colorless 5X Reaction Buffer, except GoTaq® Flexi Reaction Buffers do not contain MgCl.2. GoTaq® Flexi DNA Polymerase, on the other hand, comes with a tube containing 25 mM MgCl.2so that the reactions can be supplemented with different concentrations of magnesium.
We recommend using 1 to 1.25 units of Taq DNA polymerase in a 50 µl amplification reaction. In most cases this is an excess of enzymes and adding more enzymes will not significantly increase the yield of the product. In fact, higher amounts of enzyme increase the potential for artifacts associated with the intrinsic 5′ → 3′ exonuclease activity of Taq DNA polymerase, resulting in colored bands on the agarose gel (Longleyet al. 1990.; Bell in DeMarini, 1991).
Pipetting errors are a common cause of high enzyme levels. Small amounts of 50% glycerol enzyme solutions are difficult to dose accurately. Therefore, we strongly recommend preparing the master reaction mix, which requires a larger volume of each reagent, to reduce pipetting errors.
PCR primers define the target region to be amplified and are typically between 15 and 30 bases in length. Ideally, primers have a GC content of 40-60%. Avoid three consecutive G or C residues near the 3' end of the primer to reduce non-specific primer annealing. Also avoid primers with intramolecular or intermolecular complementary sequences to reduce primer dimer production. Intramolecular regions of secondary structure can interfere with primer-template annealing and should be avoided.
Ideally, the melting temperature (Tm), the temperature at which 50% of the primer molecules anneal to the complementary sequence, of the two primers will be within 5°C, so that the primers effectively anneal at the same temperature. Examples can be designed to include sequences useful for subsequent applications. For example, restriction enzyme sites can be placed at the 5' ends of PCR primers to facilitate subsequent cloning of the PCR product, or a T7 RNA polymerase promoter can be added to allow for in vitro transcription without the need to subcloning the PCR product into a vector.
The quality of the team
The success of amplification depends on the quantity and quality of the DNA sample. Reagents commonly used for the purification of nucleic acids (salts, guanidine, proteases, organic solvents and SDS) are strong inactivators of DNA polymerases. For example, 0.01% SDS will inhibit Taq DNA polymerase by 90%, while 0.1% SDS will inhibit Taq DNA polymerase by 99.9% (Konatet al. 1994). Some other examples of PCR inhibitors are phenol (Katcher and Schwartz, 1994), heparin (Beutleret al. 1990; coldet al. 1991), xylene cyanol, bromophenol blue (Hoppeet al. 1992), plant polysaccharides (Demeke and Adams, 1992) and polyamines spermine and spermidine (Ahokas and Erkkila, 1993). In some cases, the inhibitor does not react with the template nucleic acid. A good example of this is an inhibitor that can be released from polystyrene or polypropylene when exposed to ultraviolet light (Paoet al. 1993; Linket al. 1998).
If the amplification reaction fails and you suspect that the template DNA is contaminated with an inhibitor, add the suspected DNA preparation to a control reaction containing the template DNA and a pair of primers that have been amplified well in the past. Lack of control DNA amplification usually indicates the presence of an inhibitor. Additional steps may be required to clean up DNA preparations, such as phenol:chloroform extraction or ethanol precipitation.
The amount of template required for successful amplification depends on the complexity of the DNA sample. For example, of a 4 kb plasmid containing a 1 kb target sequence, 25% of the input DNA is the target of interest. In contrast, a 1 kb target sequence in the human genome (3.3 x 109bp) represents approximately 0.00003% of the input DNA. Therefore, approximately 1,000,000 times more human genomic DNA is required to maintain the same target copy number per reaction. Common mistakes include using too much plasmid DNA, too much PCR product, or too little genomic DNA as a template. Reactions with too little template DNA will have low yields, while reactions with too much template DNA may be affected by non-specific amplification. If possible, start with >104copies of the target sequence to obtain a signal in 25 to 30 cycles, but try to keep the final DNA concentration of the reaction ≤10 ng/µL. When re-amplifying the PCR product, the concentration of the specific PCR product is often unknown. We recommend diluting the above amplification reaction from 1:10 to 1:10,000 before re-amplification.
1 μg of 1 kb RNA = 1,77 ×12molecules
1 µg has 1 kb of bcDNA = 9.12 × 1011molecules
1 μg ADN-vector pGEM® = 2,85 × 10611molecules
1μg lambda-DNA = 1,9×1010molecules
1 μg E. coli genome-DNA = 2 × 108molecules
1 µg of human genomic DNA = 3.04 x 1065molecules
The two most frequently modified cycle parameters are annealing temperature and strain time. The durations and temperatures of the other steps in the PCR cycle usually do not differ significantly. However, in some cases the denaturation cycle can be shortened or a lower denaturation temperature can be used to reduce the number of depurination events, which can lead to mutations in the PCR products.
The order of the primers is an important factor determining the optimum annealing temperature, which is generally within 5°C of the primer's melting temperature. Using an annealing temperature slightly higher than the Tm of the primer will increase the stringency of the annealing and may minimize non-specific annealing of the primer and reduce the amount of unwanted products synthesized. Using an annealing temperature lower than the Tm of primers can result in higher yields because primers anneal more efficiently at a lower temperature. We recommend testing different annealing temperatures, starting about 5°C below Tm, to determine the best annealing conditions. In many cases, non-specific amplification and the formation of primer dimers can be reduced by optimizing the annealing temperature, but if unwanted PCR products are still a problem, consider using one of the many primer PCR methods available. to include hot use.
Oligonucleotide synthesis facilities will often provide an estimate of the Tm of the primer. Tm can also be calculated usingbiomathematical calculators. There are numerous formulas for determining the theoretical Tm of nucleic acids (Baldino, Jr.et al. 1989; Brzet al. 1990). The following formula can be used to estimate the melting temperature of oligonucleotides:
Tm = 81.5 + 16.6 × (log10[Na+]) + 0.41 × (%G+C) – 675/n
where [Na+] is the molar concentration of the salt and n = the number of bases in the oligonucleotide
An example. To calculate the melting temperature of a 22-mer oligonucleotide at 60% G+C in 50 mM KCl:
Tm = 81,5 + 16,6 × (log10[0,05]) + 0,41 × (60) – 675/22
= 81,5 + 16,6 × (–1,30) + 24,60 – 30,68 = 54 °C
The length of the elongation cycle, which may need to be optimized, depends on the size of the PCR product and the DNA polymerase used. In general, allow for approximately 1 minute per 1 kb amplicon (minimum elongation time = 1 minute) for uncorrected DNA polymerases and 2 minutes per 1 kb amplicon for corrected DNA polymerases. Avoid excessively long elongation times as they may increase the potential for artifacts related to the intrinsic exonuclease activity of 5′ → 3′ Taq DNA polymerase (Longleyet al. 1990.; Bell in DeMarini, 1991).
PCR typically involves 25 to 35 cycles of amplification. The risk of unwanted PCR products appearing in the reaction increases with an increasing number of cycles. Therefore, we recommend running only enough cycles to synthesize the desired amount of product. If non-specific amplification products accumulate before sufficient amounts of PCR product can be synthesized, consider diluting the product from the first reaction and performing a second amplification using the same primers or primers that hybridize to sequences in the PCR product . desired (nested examples) .
PCR additives and enhancers
Addition of PCR enhancers can increase the yield of the desired PCR product or reduce the production of unwanted products. There are many PCR enhancers that can operate through a number of different mechanisms. These reagents will not improve all PCRs; beneficial effects are often template and primer specific and will have to be determined empirically. Some of the most common ways of improvement are discussed below.
The addition of betaine, DMSO and formamide may be useful in amplifying GC-rich templates and templates that form strong secondary structures, which can cause DNA polymerase arrest. GC-rich templates can be problematic because of the inefficient separation of the two DNA strands or the tendency of complementary GC-rich primers to form intermolecular secondary structures, which will compete with primer-templates annealing. Betaine reduces the amount of energy required to separate DNA strands (Reeset al. 1993). It is believed that DMSO and formamide similarly promote amplification by disrupting hydrogen bond formation between the two DNA strands (Geiduschek and Herskovits, 1961).
Some reactions that amplify poorly in the absence of enhancers will yield higher yields of PCR products when betaine (1 M), DMSO (1-10%) or formamide (1-10%) is added. Concentrations of DMSO greater than 10% and formamide greater than 5% can inhibit Taq DNA polymerase and possibly other DNA polymerases (Varadaraj and Skinner, 1994).
In some cases, common stabilizers such as BSA (0.1 mg/ml), gelatin (0.1 to 1.0%) and non-ionic detergents (0 to 0.5%) can overcome amplification failure. These additives can increase the stability of DNA polymerase and reduce reagent loss by adsorption to the tube walls. BSA has also been shown to overcome the inhibitory effects of melanin in RT-PCR (Giambernardiet al. 1998). Non-ionic detergents, such as Tween®-20, NP-40 and Triton® X-100, have the added benefit of overcoming the inhibitory effects of strong ionic detergents, such as 0.01% SDS (Gelfand and White, 1990) . Ammonium ions can make the amplification reaction more tolerant of suboptimal conditions. For this reason, some PCR reagents contain 10-20 mM (NH42SO4. Other PCR enhancers include glycerol (5-20%), polyethylene glycol (5-15%) and tetramethylammonium chloride (60 mM).
Cross-contamination with nucleic acids
It is important to minimize cross-contamination between samples and prevent transfer of RNA and DNA from one experiment to another. Use separate workspaces and pipettes for pre- and post-amplification steps. Use positive displacement pipettes or aerosol-resistant tips to reduce cross-contamination during pipetting. Wear gloves and change them often.
There are several techniques that can be used to prevent the replication of contaminated DNA. PCR reagents can be treated with isopsoralen, a photoactivated cross-linking reagent that intercalates into double-stranded DNA molecules and forms interstrand covalent bonds to prevent DNA denaturation and replication. These cross-links between the strands ensure that the contaminated DNA is no longer able to replicate.
Treatment of PCR reagents with uracil-N-glycosylase (UNG), a DNA repair enzyme that hydrolyzes the base-ribose bond on uracil residues, eliminates one of the most common sources of DNA contamination: DNA products. PCR preamplified. Treatment with UNG prevents replication of uracil-containing DNA by causing DNA polymerase to linger at the resulting abasic sites. For UNG to be an effective protection against contamination, the products of previous amplifications must be synthesized in the presence of dUTP. This is easily achieved by replacing all or part of dUTP in the reaction. Non-proofreading polymerases will readily incorporate dUTP into the PCR product, but proofreading polymerases incorporate dUTP much less efficiently (Slupphauget al. 1993; Greget al.1999; I countet al. one thousand nine hundred ninety six). Because the incorporation of dUTP has no detectable effect on the intensity of ethidium bromide staining or the electrophoretic mobility of the PCR product, the reactions can be analyzed by standard agarose gel electrophoresis. While both methods are effective (Rys and Persing, 1993), UNG treatment has the advantage that both single- and double-stranded DNA templates will not be replicated (Longoet al.1990).
General considerations for RT-PCR
Also read General Considerations for PCR Optimization (above). Many of the important parameters discussed there also apply to RT-PCR. For a discussion of commonly used reverse transcriptases for RT-PCR, see the Thermostable Polymerases and Reverse Transcriptases section (below).
For RT-PCR, the success of reverse transcription depends on the integrity and purity of the RNA. Procedures for creating and maintaining a ribonuclease-free (RNase-free) environment to reduce RNA degradation are described in Blumberg, 1987. The use of an RNase inhibitor (e.g., RNasin® Recombinant Ribonuclease Inhibitor) is widely recommended. ). For optimal results, the RNA template, whether a total RNA preparation, an mRNA population or a synthesized RNA transcript, should be free of DNA to avoid amplification of contaminating DNA. The most commonly used DNA polymerases for PCR do not have reverse transcriptase activity under standard reaction conditions and therefore amplification products will only be generated if the template contains trace amounts of DNA with similar sequences.
The success of RT-PCR also depends on the amount of RNA, which may need to be varied to determine the optimal amount. Excellent amplification results can be obtained with the Access and AccessQuick™ RT-PCR systems using total RNA sample levels ranging from 1 pg to 1 µg per reaction (Figure 3) or poly(A) RNA sample levels. + in the range from 1 hp to 100 ng. .
Design of a reverse transcription primer
The selection of an appropriate primer for reverse transcription depends on the size of the target mRNA and the presence of a secondary structure. For example, a primer that hybridizes specifically to the 3' end of the transcript (a sequence-specific primer or oligo(dT) primer) can be problematic when reverse transcribing the 5' ends of long mRNAs or molecules that have important secondary properties . structure, which can cause reverse transcriptase to stall during cDNA synthesis. Random hexamers are primed for reverse transcription at multiple points along the transcript. For this reason, they are useful for both long mRNAs and transcripts with significant secondary structure.
Where possible, we recommend using primers that hybridize only to defined sequences in specific RNAs (sequence-specific primers) rather than to the entire RNA population in the sample (e.g., random hexamers or oligo(dT) primers). To distinguish between amplification of cDNA and amplification of contaminant genomic DNA, primers must be designed to hybridize to sequences in exons on either side of the intron so that any amplification product derived from genomic DNA is much larger than the product amplified from the intronic target cDNA. This size difference not only allows the two products to be distinguished by gel electrophoresis, but also promotes the synthesis of a smaller cDNA-derived product (amplification of smaller fragments is usually more efficient than that of long fragments).
Regardless of the choice of primer, the final concentration of the primer in the reaction is usually in the range of 0.1 to 1.0 µM, but may need to be optimized. We recommend using a final primer concentration of 1 µM (50 pmol in a 50 µL reaction) as a starting point for optimization. More information on PCR primer design is available in the PCR Primer Design section.
Efficient first strand cDNA synthesis can be achieved in a 20-60 minute incubation at 37-45°C using AMV reverse transcriptase or at 37-42°C for M-MLV reverse transcriptase. When using AMV RT, we recommend using a sequence-specific primer as a starting point and performing reverse transcription at 45°C for 45 minutes. A higher reaction temperature will minimize the effects of RNA secondary structure and promote full-length cDNA synthesis. First strand cDNA synthesis with random hexamers and oligo(dT) primer should be performed at room temperature (20–25°C), i.e. 37°C.
The Access and AccessQuick™ RT-PCR systems do not require RNA denaturation before starting the reverse transcription reaction. However, if desired, a denaturation step can be performed by incubating a separate tube containing the primers and template RNA at 94°C for 2 minutes. Do not incubate AMV reverse transcriptase at 94°C; will be turned off. The template/primer mix can then be cooled to 45°C and added to the RT-PCR mix for a standard reverse transcription incubation at 45°C. Following reverse transcription, we recommend a 2 minute incubation at 94°C to denature the RNA/cDNA hybrid, inactivate AMV reverse transcriptase and separate AMV RT from cDNA. It has been reported that AMV reverse transcriptase must be inactivated to obtain high yields of amplification products (Sellneret al. 1992.; Tsjoemakov, 1994).
Most RNA samples can be detected in 30 to 40 amplification cycles. If the target RNA is rare or only a small amount of starting material is available, it may be necessary to increase the number of cycles to 45 or 50 or to dilute and re-amplify the products of the first reaction.
Thermostabiele polymerasen en reverse transcriptasen
Thermostable DNA polymerase
Before using thermostable DNA polymerases in PCR, researchers had to painstakingly supplement the reaction with fresh enzyme (such as Klenow or T4 DNA polymerase) after each denaturation cycle. Thermostable DNA polymerases revolutionized and popularized PCR for their ability to withstand high denaturation temperatures. The use of thermostable DNA polymerases also allowed higher annealing temperatures, which improved the stringency of the primer annealing.
Thermostable DNA polymerases can be used for single or dual enzyme RT-PCR (Myers and Gelfand, 1991; Chiocchia and Smith, 1997). For example, Tth DNA polymerase can act as a reverse transcriptase in the presence of Mn.2+for RT-PCR of the enzyme (Myers and Gelfand, 1991). All of the DNA polymerases listed below can be used to amplify the first strand cDNA produced by a reverse transcriptase, such as AMV RT, in two-enzyme RT-PCR.
Thermostable DNA polymerases can be divided into two groups: those with 3′ → 5′ (proofreading) exonuclease activity, such as Pfu DNA polymerase, and those without proofreading function, such as Taq DNA polymerase. These two groups have some important differences. Proofreading DNA polymerases are more accurate than non-proofreading polymerases because of their 3′ → 5′ exonuclease activity, which can remove the misincorporated nucleotide from the growing DNA strand. When the amplified product needs to be cloned, expressed or used in mutational analysis, Pfu DNA polymerase is the better choice due to its high fidelity. However, for routine PCR, where the goal is simply to detect the amplification product, Taq DNA polymerase is the enzyme of choice, as yields are typically higher with unscreened DNA polymerase measurements.
Amplification with non-proofreading DNA polymerases results in the independent addition of a single nucleotide template to the 3' end of the PCR product, while the use of proofreading DNA polymerases results in blunt-ended PCR products (Clark, 1988; Hu, 1993). . More than a single nucleotide can simplify cloning of PCR products.
DNA polymerase proofreaders are also used in mixtures with uncorrected DNA polymerases, or amino-terminally truncated versions of Taq DNA polymerase, to amplify longer stretches of DNA with greater accuracy than uncorrected DNA polymerase alone (Barnes, 1994; Clineet al. 1996).
DNA polymerase was isolated from Taqwater thermosand catalyzes primer-dependent incorporation of nucleotides into duplex DNA in the 5′ → 3′ direction in the presence of Mg2+. The enzyme has no 3' → 5' exonuclease activity, but does have 5' → 3' exonuclease activity.
Taq DNA polymerase is suitable for most PCR applications that do not require a high-fidelity enzyme, such as the detection of specific DNA or RNA sequences. The error rate of Taq DNA polymerase is about 1 x 10–5errors/base, although reliability depends somewhat on reaction conditions. Accuracy is slightly higher at lower pH, lower magnesium concentration and relatively low dNTP concentration (Eckert and Kunkel, 1990; Eckert and Kunkel, 1991).
Taq DNA polymerase is commonly used to amplify PCR products of 5 kb or less. PCR products in the 5-10 kb range can be amplified with Taq DNA polymerase, but often require more optimization than smaller PCR products. For products larger than about 10 kb, we recommend an enzyme or enzyme mixture and reaction conditions designed for extensive PCR.
Taq DNA polymerase is a processive enzyme with an expansion rate of >60 nucleotides/second at 70°C (Inniset al. 1988), so a 1 minute extension step per 1 kb to be amplified should be sufficient to generate a full-length PCR product. The enzyme has a half-life of 40 minutes at 95°C (lawyeret al. 1993). Because Taq DNA polymerase is a non-reading polymerase, PCR products generated with Taq DNA polymerase will contain a 3' overhang of a single nucleotide, usually a 3' A overhang.
Tfl DNA polymerase catalyzes primer-dependent nucleotide polymerization in duplex DNA in the presence of Mg2+. In the presence of M2+, Tfl DNA polymerase can use RNA as a template. Tfl DNA polymerase shows 5' → 3' exonuclease activity but lacks 3' → 5' exonuclease activity. This enzyme is commonly used in PCR (Gaensslenet al. 1992), where its activity is comparable to that of Taq DNA polymerase. Tfl DNA polymerase is used in the Access and AccessQuick™ RT-PCR systems.
Tth DNA polymerase catalyzes the polymerization of nucleotides into duplex DNA in the 5′ → 3′ direction in the presence of MgCl2. The enzyme can use the RNA template in the presence of MnCl2(Myers and Gelfand, 1991; Ruttimannet al. 1985). Tth DNA polymerase shows 5' → 3' exonuclease activity but no detectable 3' → 5' exonuclease activity. The error rate of Tth DNA polymerase was measured as 7.7 x 10 6–5errors/base (arakawaet al. one thousand nine hundred ninety six). Tth DNA polymerase can amplify target DNA in the presence of phenol-saturated buffer (Katcher and Schwartz, 1994) and has been reported to be more resistant to inhibition by blood components than other thermostable polymerases (Ehrlichet al. 1991.; Bej i Mahbubani, 1992).
The DNA polymerase is commonly used for PCR (Myers and Gelfand, 1991; Carballeiraet al. 1990) and RT-PCR (Myers and Gelfand, 1991; Chiocchia and Smith, 1997). For primer extension, RT-PCR and cDNA synthesis using RNA templates with complex secondary structure, the high reaction temperature of Tth DNA polymerase may be an advantage over more commonly used reverse transcriptases such as AMV and M reverse transcriptases. - MLV. Recombinant Tth DNA polymerase has been shown to exhibit activity similar to that of RNase H (Aueret al. 1995).
Pfu DNA polymerase has one of the lowest error rates of all known thermophilic DNA polymerases used for amplification due to its high 3′ → 5′ exonuclease activity (Clineet al. 1996.; Andréet al. 1997). For DNA cloning and expression after PCR, Pfu DNA polymerase is usually the enzyme of choice. Pfu DNA polymerase can only be used to amplify DNA fragments up to 5 kb by extending the elongation time to 2 minutes per kilobase. It is also used in mixtures with non-proofreading DNA polymerases, such as Taq DNA polymerase, to obtain longer amplification products than with Pfu DNA polymerase alone (Barnes, 1994). However, proofreading activity can shorten PCR primers, resulting in decreased performance and increased non-specific amplification.
The discovery of reverse transcriptases, or RNA-dependent DNA polymerases, and their role in retroviral infections (Baltimore, 1970; Temin and Mizutani, 1970) disrupted the central dogma of DNA → RNA → protein molecular biology. Reverse transcriptases use an RNA template for DNA synthesis and require a primer for synthesis, just like other DNA polymerases. For in vitro applications, the primer can be oligo(dT), which hybridizes to the poly(A)+ tails of eukaryotic mRNAs, random hexamers, which drive synthesis along an RNA template, or an RNA-specific primer. which hybridizes to a known sequence within the RNA template. Subsequently, polymerization from the primer continues as in DNA-dependent DNA polymerases. The commonly used reverse transcriptases, AMV reverse transcriptase, M-MLV reverse transcriptase, and M-MLV reverse transcriptase, RNase H minus, perform the same reaction but at different optimal temperatures (AMV, 42°C; M-MLV, 37°C). and M-MLV RT, RNase H–, 42°C).
Some reverse transcriptases also possess intrinsic 3' or 5' exoribonuclease (RNase) activity, which is generally used to degrade the RNA template following first strand cDNA synthesis. Absence of 5' exoribonuclease (RNase H) activity may aid production of longer cDNAs (Bergeret al. 1983).
Some DNA-dependent DNA polymerases also possess reverse transcriptase activity, which can be enhanced under certain circumstances. For example, the thermostable and DNA-dependent Tth DNA polymerase exhibits reverse transcriptase activity when Mn2+is replaced by mg2+at the reaction.
AMV RT catalyzes DNA polymerization using DNA, RNA, or RNA:DNA hybrid templates (Houtset al. 1979). AMV reverse transcriptase is the preferred reverse transcriptase for high secondary structure templates due to its higher reaction temperature (up to 58°C). AMV RT is used in a variety of applications, including cDNA synthesis (Houtset al. 1979; Gubler and Hoffman, 1983), RT-PCR and rapid amplification of cDNA ends (RACE; Skinneret al. 1994). Although its high temperature optimum (42°C) makes it the enzyme of choice for cDNA synthesis using templates with a complex secondary structure, its relatively high RNase H activity limits its utility for generating long (>5kb) cDNAs. For these templates, M-MLV RT or M-MLV RT, RNase H minus, may be a better choice.
M-MLV RT is a single polypeptide RNA-dependent DNA polymerase. The enzyme also has DNA-dependent DNA polymerase activity at elevated enzyme levels (Rothet al.1985). M-MLV RT is used in a variety of applications, including cDNA synthesis, RT-PCR and RACE (Gerard, 1983). The relatively low RNase H activity compared to AMV RT makes M-MLV RT the enzyme of choice for generating long (>5 kb) cDNAs (Sambrook and Russell, 2001). However, for short templates with complex secondary structure, AMV RT or M-MLV RT, RNase H minus, may be better choices due to their higher optimal temperatures. M-MLV RT is processed less than AMV RT, so more units of M-MLV RT may be needed to generate the same amount of cDNA (Schaefer, 1995).
M-MLV reverse transcriptase, minus RNase H
M-MLV reverse transcriptase, RNase H minus, is a 5′ → 3′ RNA-dependent DNA polymerase that has been genetically altered to eliminate the associated ribonuclease H activity, resulting in RNA strand degradation of the RNA:DNA hybrid (Tanese and Goff, 1988). The absence of RNase H activity makes M-MLV, RNase H minus, the enzyme of choice for generating long (>5 kb) cDNAs. However, for shorter templates with a complex secondary structure, AMV reverse transcriptase may be a better option as it can be used at higher reaction temperatures.
There are two forms of M-MLV, RNase H minus: a deletion mutant and a point mutant. As the names suggest, the deletion mutant has a specific sequence deleted in the RNase H domain, and the point mutant has an introduced point mutation in the RNase H domain, while the native M-MLV RT has a recommended reaction temperature of 37 °C. the point and deletion mutants are more stable at higher temperatures and can be used at reaction temperatures up to 50°C or 55°C, depending on the reverse transcription primers used. The point mutant is often preferred over the deletion mutant because the point mutant has DNA polymerase activity similar to that of the wild-type M-MLV enzyme, while the deletion mutant has slightly reduced DNA polymerase activity compared to that of the the wild-type enzyme. (Figure 4). ).
Thermostable polymerases and reverse transcriptase products
GoTaq® G2 DNA Polymerase is a full-length recombinant Taq polymerase supplied with buffers designed for enhanced amplification.
Reverse transcriptase products include AMV, M-MLV and GoScript, an optimized formulation of M-MLV that provides robust and reliable cDNA synthesis from a full range of rare and abundant transcripts.
See Taq products and accessoriesSee reverse transcriptases and accessories
Examples of protocols
Definitive PCR Protocol Example: GoTaq® G2 DNA Polymerase
- Reaction Buffer and GoTaq® G2 DNA Polymerase (Catalog Number M7841)
- PCR-nucleotidemix (cat.nr. C1141)
- Water without nuclear energy (cat.br. P1193)
- upstream primer
- downstream primer
- DNA template
- mineral oil (optional)
- Mix the following components on ice in a sterile nuclease-free microcentrifuge tube:
- If using a thermal cycler without a heated lid, cover the reaction mixture with 1-2 drops (approximately 50 µl) of mineral oil to prevent evaporation during the thermal cycle. Centrifuge the reactions for 5 seconds in a microfuge.
- Place the reactions in a thermal cycler heated to 94-95 °C and run the PCR.
GoTaq® green or colorless 5X reaction buffer1
1X (1,5 mM MgCl2)2
PCR-nucleotidenmengsel, 10 mM
0,2 mm slinger dNTP
GoTaq® G2 DNA-polymerase (5u/μl)
<0.5 µg/50 µl
1Thaw completely and shake well before use.
2More MgCl2can be added to the reaction using 25 mM MgCl2Solution (cat. no. A3511)
Example RT protocol: First strand cDNA synthesis
The following procedure can be used to convert up to 5 µg of total RNA or up to 500 ng of poly(A) RNA to first strand cDNA.
- GoScript® Reverse Transcription System (cat. no. M5000)
- high quality experimental RNA
- Mix and centrifuge each component shortly before use. Combine the following:
- Xµl experimental RNA (up to 5µg/reaction)
- Cebador [Oligo(dT)15(0.5 µg/reaction) and/or random primer (0.5 µg/reaction) or gene specific primer (10-20 pmol/reaction)]
- Xµl nuclease free water to a final volume of 5µl
- Heat in a thermoblock for 5 minutes at 70 °C. Immediately cool in ice water for at least 5 minutes. Centrifuge for 10 seconds in a microcentrifuge. Keep on ice until the reverse transcription mixture is added.
- Prepare the reverse transcription reaction mix, 15 µl for each cDNA reaction. Mix on ice in the order listed.
- GoScript™ 5X reactiebuffer 4,0 µl
- 1,2–6,4 µl MgCl2(final concentration 1.5–5.0 mM)1
- 1.0 µl PCR nucleotide mix (final concentration 0.5 mM each dNTP)2
- 20 Units of RNasin® Recombinant Ribonuclease Inhibitor (optional)
- 1.0 µl GoScript™ reverse transcriptase
- Xμl Nuclease Free Water (to a final volume of 15 μl)
1magnesium2+The concentration should be optimized to 1.5–5.0 mM (MgCl2obtained at 25 mM).
2If isotopic or non-isotopic incorporation is desired to monitor first-strand cDNA synthesis, α[32P]-dCTP or other modified nucleotides can be complemented in the PCR nucleotide mixture.
Reactions can be stopped at this point for cDNA analysis or frozen for long-term storage.
Related PCR products and resources
- Taq-polymerase in point-PCR
- PCR cloning
- qPCR en RT-qPCR
- GoTaq® G2 Hot Start Polymer
- GoTaq® G2 Hot Start Master microfoonsevi
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Introduction to PCR qPCR en RT-qPCR General Considerations for PCR Optimization General considerations for RT-PCR Thermostabiele polymerasen en reverse transcriptasen Examples of protocols Related PCR products and resources Reference