The dye is simply added to the reaction, and fluorescence is measured at each PCR cycle. Because fluorescence of these dyes increases dramatically in the presence of double-stranded DNA, DNA synthesis can be monitored as an increase in fluorescent signal. However, preliminary work often must be done to ensure that the PCR conditions yield only specific product. In subsequent reactions, specific amplification can verified by a melt curve analysis.
The product length and sequence affect melting temperature Tm , so the melt curve is used to characterize amplicon homogeneity. Nonspecific amplification can be identified by broad peaks in the melt curve or peaks with unexpected Tm values. By distinguishing specific and nonspecific amplification products, the melt curve adds a quality control aspect during routine use. These probes also can be used to detect single nucleotide polymorphisms Lee et al.
There are several general categories of real-time PCR probes, including hydrolysis, hairpin and simple hybridization probes. These probes contain a complementary sequence that allows the probe to anneal to the accumulating PCR product, but probes can differ in the number and location of the fluorescent reporters.
During the annealing step, the probe hybridizes to the PCR product generated in previous amplification cycles. The fluor is freed from the effects of the energy-absorbing quencher, and the progress of the reaction and accumulation of PCR product is monitored by the resulting increase in fluorescence.
With this approach, preliminary experiments must be performed prior to the quantitation experiments to show that the signal generated is proportional to the amount of the desired PCR product and that nonspecific amplification does not occur. Hairpin probes, also known as molecular beacons, contain inverted repeats separated by a sequence complementary to the target DNA. The hairpin probe is designed so that the probe binds preferentially to the target DNA rather than retains the hairpin structure.
As the reaction progresses, increasing amounts of the probe anneal to the accumulating PCR product, and as a result, the fluor and quencher become physically separated. The fluor is no longer quenched, and the level of fluorescence increases. One advantage of this technique is that hairpin probes are less likely to mismatch than hydrolysis probes Tyagi et al. However, preliminary experiments must be performed to show that the signal is specific for the desired PCR product and that nonspecific amplification does not occur.
The use of simple hybridization probes involves two labeled probes or, alternatively, one labeled probe and a labeled PCR primer. In the first approach, the energy emitted by the fluor on one probe is absorbed by a fluor on the second probe, which hybridizes nearby.
In the second approach, the emitted energy is absorbed by a second fluor that is incorporated into the PCR product as part of the primer. Both of these approaches result in increased fluorescence of the energy acceptor and decreased fluorescence of the energy donor. The use of hybridization probes can be simplified even further so that only one labeled probe is required.
In this approach, quenching of the fluor by deoxyguanosine is used to bring about a change in fluorescence Crockett and Wittwer, ; Kurata et al. The labeled probe anneals so that the fluor is in close proximity to G residues within the target sequence, and as probe annealing increases, fluorescence decreases due to deoxyguanosine quenching. With this approach, the location of probe is limited because the probe must hybridize so that the fluorescent dye is very near a G residue.
The advantage of simple hybridization probes is their ability to be multiplexed more easily than hydrolysis and hairpin probes through the use of differently colored fluors and probes with different melting temperatures reviewed in Wittwer et al. Many of these suggestions also apply when using other DNA polymerases.
Magnesium is a required cofactor for thermostable DNA polymerases, and magnesium concentration is a crucial factor that can affect amplification success. Template DNA concentration, chelating agents present in the sample e. In the absence of adequate free magnesium, Taq DNA polymerase is inactive. Excess free magnesium reduces enzyme fidelity Eckert and Kunkel, and may increase the level of nonspecific amplification Williams, ; Ellsworth et al.
For these reasons, researchers should empirically determine the optimal magnesium concentration for each target. To do so, set up a series of reactions containing 1. The effect of magnesium concentration and the optimal concentration range can vary with the particular DNA polymerase. For example, the performance of Pfu DNA polymerase seems depend less on magnesium concentration, but when optimization is required, the optimal concentration is usually in the range of 2—6mM.
Before assembling the reactions, be sure to thaw the magnesium solution completely prior to use and vortex the magnesium solution for several seconds before pipetting.
Magnesium chloride solutions can form concentration gradients as a result of multiple freeze-thaw cycles, and vortex mixing is required to obtain a uniform solution. These two steps, though seemingly simple, eliminate the cause of many failed experiments. Some scientists prefer to use reaction buffers that already contain MgCl 2 at a final concentration of 1. It should be noted, however, that Hu et al. The free magnesium changes of 0.
They postulated that magnesium chloride precipitates as a result of multiple freeze-thaw cycles. Figure 2. Effects of magnesium concentration on amplification. Amplifications were performed using various Mg concentrations to demonstrate the effect on the amplification of a 1. The reaction products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining.
Most reaction buffers consist of a buffering agent, most often a Tris-based buffer, and salt, commonly KCl. The buffer regulates the pH of the reaction, which affects DNA polymerase activity and fidelity. The buffer also contains a compound that increases the density of the sample so that it will sink into the well of the agarose gel, allowing reactions to be directly loaded onto an agarose gel without the need for loading dye. Both buffers are supplied at pH 8. We recommend using 1—1. In most cases, this is an excess of enzyme, and adding more enzyme will not significantly increase product yield.
Pipetting errors are a frequent cause of excessive enzyme levels. PCR primers define the target region to be amplified and generally range in length from 15—30 bases. Also, avoid primers with intra- or intermolecular complementary sequences to minimize the production of primer-dimer.
Intramolecular regions of secondary structure can interfere with primer annealing to the template and should be avoided. Primers can be designed to include sequences that are useful for downstream applications. Successful amplification depends on DNA template quantity and quality.
Reagents commonly used to purify nucleic acids salts, guanidine, proteases, organic solvents and SDS are potent inactivators of DNA polymerases. For example, 0. In some cases, the inhibitor is not introduced into the reaction with the nucleic acid template. A good example of this is an inhibitory substance that can be released from polystyrene or polypropylene upon exposure to ultraviolet light Pao et al.
If an amplification reaction fails and you suspect the DNA template is contaminated with an inhibitor, add the suspect DNA preparation to a control reaction with a DNA template and primer pair that has amplified well in the past.
Failure to amplify the control DNA usually indicates the presence of an inhibitor. Additional steps to clean up the DNA preparation, such as phenol:chloroform extraction or ethanol precipitation, may be necessary. The amount of template required for successful amplification depends upon the complexity of the DNA sample.
Conversely, a 1kb target sequence in the human genome 3. Thus, approximately 1,,fold more human genomic DNA is required to maintain the same number of target copies per reaction.
Reactions with too little DNA template will have low yields, while reactions with too much DNA template can be plagued by nonspecific amplification. We recommend diluting the previous amplification reaction to , before reamplification. The two most commonly altered cycling parameters are annealing temperature and extension time.
The lengths and temperatures for the other steps of a PCR cycle do not usually vary significantly. However in some cases, the denaturation cycle can be shortened or a lower denaturation temperature used to reduce the number of depurination events, which can lead to mutations in the PCR products. Using an annealing temperature slightly higher than the primer Tm will increase annealing stringency and can minimize nonspecific primer annealing and decrease the amount of undesired products synthesized.
Using an annealing temperature lower than the primer Tm can result in higher yields, as the primers anneal more efficiently at the lower temperature. In many cases, nonspecific amplification and primer-dimer formation can be reduced through optimization of annealing temperature, but if undesirable PCR products remain a problem, consider incorporating one of the many hot-start PCR methods.
Oligonucleotide synthesis facilities will often provide an estimate of a primer's Tm. The Tm also can be calculated using the Biomath Calculators. Numerous formulas exist to determine the theoretical Tm of nucleic acids Baldino, Jr.
The formula below can be used to estimate the melting temperature for oligonucleotides:. The length of the extension cycle, which may need to be optimized, depends on PCR product size and the DNA polymerase being used. PCR typically involves 25—35 cycles of amplification. The risk of undesirable PCR products appearing in the reaction increases as the cycle number increases, so we recommend performing only enough cycles to synthesize the desired amount of product.
If nonspecific amplification products accumulate before sufficient amounts of PCR product can be synthesized, consider diluting the products of the first reaction and performing a second amplification with the same primers or primers that anneal to sequences within the desired PCR product nested primers. Addition of PCR-enhancing agents can increase yield of the desired PCR product or decrease production of undesired products.
There are many PCR enhancers, which can act through a number of different mechanisms. These reagents will not enhance all PCRs; the beneficial effects are often template- and primer-specific and will need to be determined empirically. Some of the more common enhancing agents are discussed below.
Addition of betaine, DMSO and formamide can be helpful when amplifying GC-rich templates and templates that form strong secondary structures, which can cause DNA polymerases to stall.
GC-rich templates can be problematic due to inefficient separation of the two DNA strands or the tendency for the complementary, GC-rich primers to form intermolecular secondary structures, which will compete with primer annealing to the template.
Betaine reduces the amount of energy required to separate DNA strands Rees et al. DMSO and formamide are thought to aid amplification in a similar manner by interfering with hydrogen bond formation between two DNA strands Geiduschek and Herskovits, In some cases, general stabilizing agents such as BSA 0.
These additives can increase DNA polymerase stability and reduce the loss of reagents through adsorption to tube walls. Ammonium ions can make an amplification reaction more tolerant of nonoptimal conditions.
It is important to minimize cross-contamination between samples and prevent carryover of RNA and DNA from one experiment to the next. Use separate work areas and pipettors 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 a number of techniques that can be used to prevent amplification of contaminating DNA. PCR reagents can be treated with isopsoralen, a photo-activated, cross-linking reagent that intercalates into double-stranded DNA molecules and forms covalent, interstrand crosslinks, to prevent DNA denaturation and replication.
These inter-strand crosslinks effectively render contaminating DNA unamplifiable. For UNG to be an effective safeguard against contamination, the products of previous amplifications must be synthesized in the presence of dUTP. Since dUTP incorporation has no noticeable effect on the intensity of ethidium bromide staining or electrophoretic mobility of the PCR product, reactions can be analyzed by standard agarose gel electrophoresis.
While both methods are effective Rys and Persing, , UNG treatment has the advantage that both single-stranded and double-stranded DNA templates will be rendered unamplifiable Longo et al.
Procedures for creating and maintaining a ribonuclease-free RNase-free environment to minimize RNA degradation are described in Blumberg, The use of an RNase inhibitor e.
The most commonly used DNA polymerases for PCR have no reverse transcriptase activity under standard reaction conditions, and thus, amplification products will be generated only if the template contains trace amounts of DNA with similar sequences. Figure 3. Amplification of a specific message in total RNA. The specific bp amplicon is indicated. Selection of an appropriate primer for reverse transcription depends on target mRNA size and the presence of secondary structure.
Random hexamers prime reverse transcription at multiple points along the transcript. For this reason, they are useful for either long mRNAs or transcripts with significant secondary structure.
Whenever possible, we recommend using a primer that anneals only to defined sequences in particular RNAs sequence-specific primers rather than to entire RNA populations in the sample e.
To differentiate between amplification of cDNA and amplification of contaminating genomic DNA, design primers to anneal to sequences in exons on opposite sides of an intron so that any amplification product derived from genomic DNA will be much larger than the product amplified from the target cDNA. This size difference not only makes it possible to differentiate the two products by gel electrophoresis but also favors the synthesis of the smaller cDNA-derived product amplification of smaller fragments is often more efficient than that of long fragments.
Regardless of primer choice, the final primer concentration in the reaction is usually within the range of 0. The higher reaction temperature will minimize the effects of RNA secondary structure and encourage full-length cDNA synthesis.
It has been reported that AMV reverse transcriptase must be inactivated to obtain high yields of amplification product Sellner et al. Most RNA samples can be detected using 30—40 cycles of amplification.
If the target RNA is rare or if only a small amount of starting material is available, it may be necessary to increase the number of cycles to 45 or 50 or dilute the products of the first reaction and reamplify. Thermostable DNA polymerases revolutionized and popularized PCR because of their ability to withstand the high denaturation temperatures.
The use of thermostable DNA polymerases also allowed higher annealing temperatures, which improved the stringency of primer annealing. These two groups have some important differences. When the amplified product is to be cloned, expressed or used in mutation analysis, Pfu DNA polymerase is a better choice due to its high fidelity.
However, for routine PCR, where simple detection of an amplification product is the goal, Taq DNA polymerase is the most commonly used enzyme because yields tend to be higher with a nonproofreading DNA polymerase. The single-nucleotide overhang can simplify the cloning of PCR products.
The fidelity is slightly higher at lower pH, lower magnesium concentration and relatively low dNTP concentration Eckert and Kunkel, ; Eckert and Kunkel, For products larger than approximately 10kb, we recommend an enzyme or enzyme mix and reaction conditions that are designed for long PCR.
This enzyme is commonly used in PCR Gaensslen et al. The error rate of Tth DNA polymerase has been measured at 7. Tth DNA polymerase can amplify target DNA in the presence of phenol-saturated buffer Katcher and Schwartz, and has been reported to be more resistant to inhibition by blood components than other thermostable polymerases Ehrlich et al.
Pfu DNA polymerase can be used alone to amplify DNA fragments up to 5kb by increasing the extension time to 2 minutes per kilobase. However, the proofreading activity can shorten PCR primers, leading to decreased yield and increased nonspecific amplification. Some DNA-dependent DNA polymerases also possess a reverse transcriptase activity, which can be favored under certain conditions. However, for shorter templates with complex secondary structure, AMV reverse transcriptase may be a better choice because it can be used at higher reaction temperatures.
As the names suggest, the deletion mutant had a specific sequence in the RNase H domain deleted, and the point mutant has a point mutation introduced in the RNase H domain. The point mutant is often preferred over the deletion mutant because the point mutant has DNA polymerase activity comparable to that of the wildtype M-MLV enzyme, whereas the deletion mutant has a slightly reduced DNA polymerase activity compared to that of the wildtype enzyme Figure 4.
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Current Password Incorrect password. New Password Minimum of 8 characters Uppercase and lowercase letters At least one number. Password doesn't meet requirements. Password has been used too recently. A similar molecule called Taq polymerase, or thermostable polymerase, is used in its stead, because it can withstand the heat requirements of PCR.
Additionally, a PCR reaction requires free nucleotides, a buffer, and magnesium. Magnesium chloride is the preferred method of adding magnesium to a PCR experiment. Thermostable polymerase requires the presence of magnesium to act as a cofactor during the reaction process. Its role is similar to that of a catalyst: the magnesium is not actually consumed in the reaction, but the reaction cannot proceed without the presence of the magnesium.
The more magnesium that is added to a PCR reaction, the quicker the reaction will proceed. However, that is not necessarily a good thing. If too much magnesium is present, the DNA polymerase will work too quickly and often make errors in the copying process. This will lead to many different strands of DNA being produced that do not necessarily represent the original sample that was provided. If magnesium is in limited supply in a reaction, it will not go as quickly as it should if at all.
You may attempt to run a 40 cycle PCR but not get the amount of copies you intended. To find an enzyme, visit our selection guide. The recommended concentration of DMSO is between 2. Some templates may have long AT-rich stretches that are hard to amplify under standard reaction conditions. The advantage of having AT-rich templates is that a lower extension temperature can be used.
DNA replication at this reduced temperature appears to be reliable Su et al. Su, X. Nucl Acids Res. Magnesium is a required cofactor for thermostable DNA polymerases and is important for successful amplification.
Salt neutralizes the negative charges on the phosphate backbone of DNA, stabilizing double-stranded DNA by offsetting negative charges that would otherwise repel one another. To improve amplification of DNA fragments, especially fragments between and 1, bp, a KCl concentration of 70— mM is recommended.
For amplification of longer products, a lower salt concentration appears to be more effective, whereas amplification of shorter products occurs optimally with higher salt concentrations. This effect is likely because high salt concentration preferentially permits denaturation of short DNA molecules over long DNA molecules.
It is important to note that a salt concentration above 50 mM can inhibit Taq polymerases. All Rights Reserved. All trademarks are the property of Takara Bio Inc. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio. As an experienced, reliable solutions provider, we offer full-scale customization services for a vast array of reagents and instruments backed by a quality management system, on-time delivery, and dependable support—giving you a competitive advantage in the ever-changing clinical landscape.
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