Although there are several T m calculators available, it is important to note that these calculations are an estimate of the actual T m due to lack of specific information about a particular reaction and assumptions made in the algorithms for the T m calculators themselves. The former will give more accurate T m estimation because it takes into account the stacking energy of neighboring base pairs.
The latter is used more frequently because the calculations are simple and can be done quickly by hand. See Troubleshooting section for information about how various PCR conditions and additives affect melting temperature. PCR thermal cyclers rapidly heat and cool the reaction mixture, allowing for heat-induced denaturation of duplex DNA strand separation , annealing of primers to the plus and minus strands of the DNA template, and elongation of the PCR product.
Any longer than 3 minutes may inactivate the DNA polymerase, destroying its enzymatic activity. One method, known as hot-start PCR, drastically extends the initial denaturation time from 3 minutes up to 9 minutes. This protocol modification avoids likely inactivation of the DNA polymerase enzyme. Refer to the Troubleshooting section of this protocol for more information about hot start PCR and other alternative methods.
The next step is to set the thermal cycler to initiate the first of 25 to 35 rounds of a three-step temperature cycle Table 2. While increasing the number of cycles above 35 will result in a greater quantity of PCR products, too many rounds often results in the enrichment of undesirable secondary products. The three temperature steps in a single cycle accomplish three tasks: the first step denatures the template and in later cycles, the amplicons as well , the second step allows optimal annealing of primers, and the third step permits the DNA polymerase to bind to the DNA template and synthesize the PCR product.
The duration and temperature of each step within a cycle may be altered to optimize production of the desired amplicon. The time for the denaturation step is kept as short as possible. Usually 10 to 60 seconds is sufficient for most DNA templates. The denaturation time and temperature may vary depending on the G-C content of the template DNA, as well as the ramp rate, which is the time it takes the thermal cycler to change from one temperature to the next.
The temperature for this step is usually the same as that used for the initial denaturation phase step 1 above; e. The cycle concludes with an elongation step. The temperature depends on the DNA polymerase selected for the experiment. Pfu DNA Polymerase is recommended for use in PCR and primer extension reactions that require high fidelity and requires 2 minutes for every 1 kb to be amplified.
See manufacturer recommendations for exact elongation temperatures and elongation time indicated for each specific DNA polymerase. The final phase of thermal cycling incorporates an extended elongation period of 5 minutes or longer. This last step allows synthesis of many uncompleted amplicons to finish and, in the case of Taq DNA polymerase, permits the addition of an adenine residue to the 3' ends of all PCR products.
This modification is mediated by the terminal transferase activity of Taq DNA polymerase and is useful for subsequent molecular cloning procedures that require a 3'-overhang. The stringency of a reaction may be modulated such that the specificity is adjusted by altering variables e. For example, if the reaction is not stringent enough, many spurious amplicons will be generated with variable lengths. If the reaction is too stringent, no product will be produced.
Troubleshooting PCR reactions may be a frustrating endeavor at times. However, careful analysis and a good understanding of the reagents used in a PCR experiment can reduce the amount of time and trials needed to obtain the desired results. However, before changing anything, be sure that an erroneous result was not due to human error.
Start by confirming all reagents were added to a given reaction and that the reagents were not contaminated. Are there non-specific products bands that migrate at a different size than the desired product?
Was there a lack of any product? Also, it is wise to analyze the G-C content of the desired amplicon. First determine if any of the PCR reagents are catastrophic to your reaction.
This can be achieved by preparing new reagents e. This process will determine which reagent was the culprit for the failed PCR experiment. In the case of very old DNA, which often accumulates inhibitors, it has been demonstrated that addition of bovine serum albumin may help alleviate the problem. Primer dimers can form when primers preferentially self anneal or anneal to the other primer in the reaction.
If this occurs, a small product of less than bp will appear on the agarose gel. Start by altering the ratio of template to primer; if the primer concentration is in extreme excess over the template concentration, then the primers will be more likely to anneal to themselves or each other over the DNA template. Adding DMSO and or using a hot start thermal cycling method may resolve the problem.
In the end it may be necessary to design new primers. Non-specific products are produced when PCR stringency is excessively low resulting in non-specific PCR bands with variable lengths.
This produces a ladder effect on an agarose gel. It then is advisable to choose PCR conditions that increase stringency.
A smear of various sizes may also result from primers designed to highly repetitive sequences when amplifying genomic DNA. However, the same primers may amplify a target sequence on a plasmid without encountering the same problem. Lack of PCR products is likely due to reaction conditions that are too stringent.
Primer dimers and hairpin loop structures that form with the primers or in the denatured template DNA may also prevent amplification of PCR products because these molecules may no longer base pair with the desired DNA counterpart.
If the G-C content has not been analyzed, it is time to do so. However, there are many additives that have been used to help alleviate the challenges. Understanding the function of reagents used on conventional PCR is critical when first deciding how best to alter reaction conditions to obtain the desired product.
However, the wrong concentration of such reagents may lead to spurious results, decreasing the stringency of the reaction. When troubleshooting PCR, only one reagent should be manipulated at a time. However, it may be prudent to titrate the manipulated reagent. Changing the magnesium concentration is one of the easiest reagents to manipulate with perhaps the greatest impact on the stringency of PCR. The 10 X PCR buffer solutions may contain 15 mM MgCl 2 , which is enough for a typical PCR reaction, or it may be added separately at a concentration optimized for a particular reaction.
If the desired amplicon is below bp and long non-specific products are forming, specificity may be improved by titrating KCl, increasing the concentration in 10 mM increments up to mM.
Thus, choosing an appropriate enzyme can be helpful for obtaining desired amplicon products. The addition of a 3' adenine has become a useful strategy for cloning PCR products into TA vectors whit 3' thymine overhangs. However, if fidelity is more important an enzyme such as Pfu may be a better choice. Several manufactures have an array of specific DNA polymerases designed for specialized needs.
Take a look at the reaction conditions and characteristics of the desired amplicon, and then match the PCR experiment with the appropriate DNA polymerase. Most manufactures have tables that aid DNA polymerase selection by listing characteristics such as fidelity, yield, speed, optimal target lengths, and whether it is useful for G-C rich amplification or hot start PCR.
Optimal target molecules are between 10 4 to 10 7 molecules and may be calculated as was described in the notes above. Additive reagents may yield results when all else fails.
Understanding the reagents and what they are used for is critical in determining which reagents may be most effective in the acquisition of the desired PCR product.
Adding reagents to the reaction is complicated by the fact that manipulation of one reagent may impact the usable concentration of another reagent. In addition to the reagents listed below, proprietary commercially available additives are available from many biotechnology companies.
Formamide final reaction concentration of 1. Formamide also has been shown to be an enhancer for G-C rich templates. As the amplicon or template DNA is denatured, it will often form secondary structures such as hairpin loops. Betaine final reaction concentration of 0. Non ionic detergents function to suppress secondary structure formation and help stabilize the DNA polymerase. Non ionic detergents such as Triton X, Tween 20, or NP may be used at reaction concentrations of 0.
The presence of non ionic detergents decreases PCR stringency, potentially leading to spurious product formation. However, their use will also neutralize the inhibitory affects of SDS, an occasional contaminant of DNA extraction protocols. Hot start PCR is a versatile modification in which the initial denaturation time is increased dramatically Table 4. This modification can be incorporated with or without other modifications to cycling conditions.
Moreover, it is often used in conjunction with additives for temperamental amplicon formation. In fact, hot start PCR is increasingly included as a regular aspect of general cycling conditions. Hot start has been demonstrated to increase amplicon yield, while increasing the specificity and fidelity of the reaction.
The rationale behind hot start PCR is to eliminate primer-dimer and non-specific priming that may result as a consequence of setting up the reaction below the T m. In general, the DNA polymerase is withheld from the reaction during the initial, elongated, denaturing time. Although other components of the reaction are sometimes omitted instead of the DNA polymerase, here we will focus on the DNA polymerase.
There are several methods which allow the DNA polymerase to remain inactive or physically separated until the initial denaturation period has completed, including the use of a solid wax barrier, anti-DNA polymerase antibodies, and accessory proteins. Alternatively, the DNA polymerase may simply be added to the reaction after the initial denaturation cycle is complete.
The concept is to design two phases of cycling conditions Table 5. The first phase employs successively lower annealing temperatures every second cycle traditionally 1.
The function of the first phase should alleviate mispriming, conferring a 4-fold advantage to the correct product.
Thus, after 10 cycles, a fold advantage would yield copies of the correct product over any spurious priming. This would allow the correct product a fold advantage over false priming products. The concept takes into account a relatively new feature associated with modern thermal cyclers, which allows adjustment of the ramp speed as well as the cooling rate.
The ramp speed is lowered to 2. Nested PCR is a powerful tool used to eliminate spurious products. The use of nested primers is particularly helpful when there are several paralogous genes in a single genome or when there is low copy number of a target sequence within a heterogeneous population of orthologous sequences.
This omission prevents the polymerase from extending primers until the critical component is added at the higher temperature where primer annealing is more stringent. However, this method is tedious and increases the risk of contamination. A second, less labor-intensive approach involves the reversible inactivation or physical separation of one or more critical components in the reaction.
The DNA polymerase also can be kept in an inactive state by binding to an oligonucleotide, also known as an aptamer Lin and Jayasena, ; Dang and Jayasena, or an antibody Scalice et al. This bond is disrupted at the higher temperatures, releasing the functional DNA polymerase.
Finally, the DNA polymerase can be maintained in an inactive state through chemical modification Moretti, T. Activity is restored during initial denaturation, allowing hot-start PCR. Amplification of long DNA fragments is desirable for numerous applications such as physical mapping applications Rose, and direct cloning from genomes.
While basic PCR works well when smaller fragments are amplified, amplification efficiency and therefore the yield of amplified fragments decreases significantly as the amplicon size increases over 5kb. This decrease in yield can be attributed to the accumulation of truncated products, which are not suitable substrates for additional cycles of amplification. These products appear as smeared, as opposed to discrete, bands on a gel.
In , Wayne Barnes Barnes, and other researchers Cheng et al. They devised an approach using a mixture of two thermostable polymerases to synthesize longer PCR products. Presumably, when the nonproofreading DNA polymerase e. The proofreading polymerase e. Although the use of two thermostable DNA polymerases can significantly increase yield, other conditions can have a significant impact on the yield of longer PCR products Cheng et al.
Logically, longer extension times can increase the yield of longer PCR products because fewer partial products are synthesized. Extension times depend on the length of the target; times of 10—20 minutes are common. In addition, template quality is crucial. Depurination of the template, which is promoted at elevated temperatures and lower pH, will result in more partial products and decreased overall yield.
In long PCR, denaturation time is reduced to 2—10 seconds to decrease depurination of the template. Additives, such as glycerol and dimethyl sulfoxide DMSO , also help lower the strand-separation and primer-annealing temperatures, alleviating some of the depurination effects of high temperatures.
Cheng et al. This optimized enzyme mixture allows efficient amplification of up to 40kb from lambda DNA or 30kb from human genomic DNA. However, a wide variety of applications, such as determining viral load, measuring responses to therapeutic agents and characterizing gene expression, would be improved by quantitative determination of target abundance. Theoretically, this should be easy to achieve, given the exponential nature of PCR, because a linear relationship exists between the number of amplification cycles and the logarithm of the number of molecules.
In practice, however, amplification efficiency is decreased because of contaminants inhibitors , competitive reactions, substrate exhaustion, polymerase inactivation and target reannealing. As the number of cycles increases, the amplification efficiency decreases, eventually resulting in a plateau effect. Normally, quantitative PCR requires that measurements be taken before the plateau phase so that the relationship between the number of cycles and molecules is relatively linear.
This point must be determined empirically for different reactions because of the numerous factors that can affect amplification efficiency. Because the measurement is taken prior to the reaction plateau, quantitative PCR uses fewer amplification cycles than basic PCR. This can cause problems in detecting the final product because there is less product to detect. To monitor amplification efficiency, many applications are designed to include an internal standard in the PCR.
Amplification of housekeeping genes verifies that the target nucleic acid and reaction components were of acceptable quality but does not account for differences in amplification efficiencies due to differences in product size or primer annealing efficiency between the internal standard and target being quantified.
In competitive PCR, a known amount of a control template is added to the reaction. This template is amplified using the same primer pair as the experimental target molecule but yields a distinguishable product e. The amounts of control and test product are compared after amplification. While these approaches control for the quality of the target nucleic acid, buffer components and primer annealing efficiencies, they have their own limitations Siebert and Larrick, ; McCulloch et al.
Numerous fluorescent and solid-phase assays exist to measure the amount of amplification product generated in each reaction, but they often fail to discriminate amplified DNA of interest from nonspecific amplification products. Some of these analyses rely on blotting techniques, which introduce another variable due to nucleic acid transfer efficiencies, while other assays were developed to eliminate the need for gel electrophoresis yet provide the requisite specificity.
Real-time PCR, which provides the ability to view the results of each amplification cycle, is a popular way of overcoming the need for analysis by electrophoresis. The use of fluorescently labeled oligonucleotide probes or primers or fluorescent DNA-binding dyes to detect and quantitate a PCR product allows quantitative PCR to be performed in real time. Specially designed instruments perform both thermal cycling to amplify the target and fluorescence detection to monitor PCR product accumulation.
DNA-binding dyes are easy to use but do not differentiate between specific and nonspecific PCR products and are not conducive to multiplex reactions.
Fluorescently labeled nucleic acid probes have the advantage that they react with only specific PCR products, but they can be expensive and difficult to design. 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. These two developments let to the automation of PCR. A representative temperature profile for each cycle might look like the following:. We could design an oligonucleotide with two Ahd I restriction sequences, with slightly different sequences in the interrupted region of the palindrome, to give:.
If this this were inserted into a vector, and the vector then was cut with Ahd I, it would have the following sequence at the ends of the linearized vector:. In other words, a 3' 'T' overhang at both ends of the vector.
Thus, two main advances allowed the process to be automated , these advances were: The use of thermostable DNA polymerases , which resisted denaturation inactivation at high temperatures. Thus, an initial aliquot of polymerase could last throughout the numerous cycles of the protocol. The first thermostable DNA polymerase to be used was isolated from the bacterium Thermus aquaticus. It was isolated from a hot spring in Yellowstone National Park where it lived happily i.
These are known as thermal cyclers or PCR machines. Thermal cycling parameters The thermal cycling parameters are critical to a successful PCR experiment. The important steps in each cycles of PCR include: denaturation of template annealing of primers extension of the primers A representative temperature profile for each cycle might look like the following: Figure 3. Supercoiled plasmids are tougher to melt and may require boiling for several minutes, or may be initially denatured by using base NaOH, followed by pH neutralization.
Denaturation during the PCR experiment i. Primer annealing temperature Primer annealing temperature is an important parameter in the success of the PCR experiment. The annealing temperature is characteristic for each oligonucleotide: it is a function of the length and base composition of the primer as well as the ionic strength of the reaction buffer. Estimates of the annealing temperature can be calculated in several different ways. These calculated annealing temperatures are a starting point for the PCR experiment, but ideal annealing temperatures are determined empirically.
The length of time of the primer extension steps can be increased if the region of DNA to be amplified is long, however, for the majority of PCR experiments an extension time of 2 minutes is sufficient to get complete extension. Number of cycles The number of cycles is usually between 25 and More cycles mean a greater yield of product.
However, with increasing number of cycles the greater the probability of generating various artifacts e. It is unusual to find procedures which have more than 40 cycles. RT activity Weak Weak? Primers Primer design Generally, primers used are 20 - 30 mer in length. This provides for practical annealing temperatures of the high temperature regimen where the thermostable polymerase is most active.
Primers should avoid stretches of polybase sequences e. Inverted repeat sequences should be avoided so as to prevent formation of secondary structure in the primer, which would prevent hybridization to template Sequences complementary to other primers used in the PCR should be avoid so as to prevent hybridization between primers particularly important for the 3' end of the primer If possible the 3' end of the primer should be rich in G, C bases to enhance annealing of the end which will be extended The distance between primers should be less than 10 Kb in length.
Melting temperature Tm of primers The Tm of primer hybridization can be calculated using various formulas. This formula is reportedly useful for primers of 14 to 70 bases in length.
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