advantage gc 2 pcr user manual

Once assembled, the reaction is placed in a thermal cycler, an instrument that subjects the reaction to a series of different temperatures for set amounts of time. This series of temperature and time adjustments is referred to as one cycle of amplification. Each PCR cycle theoretically doubles the amount of targeted sequence (amplicon) in the reaction. Ten cycles theoretically multiply the amplicon by a factor of about one thousand; 20 cycles, by a factor of more than a million in a matter of hours. In the denaturation process, the two intertwined strands of DNA separate from one another, producing the necessary single-stranded DNA template for replication by the thermostable DNA polymerase. Finally, the synthesis of new DNA begins as the reaction temperature is raised to the optimum for the DNA polymerase.If the temperature during the annealing and extension steps are similar, these two steps can be combined into a single step in which both primer annealing and extension take place.Yet numerous instances exist in which amplification of RNA would be preferred. To apply PCR to the study of RNA, the RNA sample must first be converted to cDNA to provide the necessary DNA template for the thermostable polymerase (Figure 1). This process is called reverse transcription (RT), hence the name RT-PCR. Reverse transcriptases have been adapted for use in a variety of in vitro applications including real-time and endpoint RT-PCR, labeled-cDNA probe generation and cDNA library construction. The ideal reverse transcriptase is robust (highly active under a variety of conditions) and converts all primed RNA within a sample to cDNA, regardless of its abundance, length or secondary structure. Genetic engineering and development of proprietary RT-enhancing buffers have led to the commercial availability of new enzymes that offer superior performance over these naturally occurring RTs. Some thermostable DNA polymerases (e.g.

• advantage gc 2 pcr user manual, advantage gc 2 pcr user manual pdf, advantage gc 2 pcr user manual download, advantage gc 2 pcr user manual free, advantage gc 2 pcr user manual online.

Your commerce experience may be limited. Please update your browser to Internet Explorer 11 or above. When you select your country, you agree that we can place these functional cookies on your device. After that, you will need to contact Customer Service to unlock your account. Please try again or contact Customer Service. Please request another reset link. Please try again or contact Customer Service. A verified email address is required to access the full functionality of your Promega.com account. Please try again or contact Customer Service. Please try again or contact Customer Service. Please try again or contact Customer Service. Please check your network settings and try again. Please try again or contact Customer Service. 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 amplification of DNA sequences by PCR requires only hours. While most biochemical analyses, including nucleic acid detection with radioisotopes, require the input of significant amounts of biological material, the PCR process requires very little. Thus, PCR can achieve more sensitive detection and higher levels of amplification of specific sequences in less time than previously used methods. These features make the technique extremely useful, not only in basic research, but also in commercial uses, including genetic identity testing, forensics, industrial quality control and in vitro diagnostics. Basic PCR is commonplace in many molecular biology labs where it is used to amplify DNA fragments and detect DNA or RNA sequences within a cell or 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 different types of PCR methods, applications and optimization.

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. They devised an approach using a mixture of two thermostable polymerases to synthesize longer PCR products. Presumably, when the nonproofreading DNA polymerase (e.g., Taq DNA polymerase) misincorporates a dNTP, subsequent extension of the newly synthesized DNA either proceeds very slowly or stops completely. The proofreading polymerase (e.g., Pfu DNA polymerase or Tli DNA polymerase) serves to remove the misincorporated nucleotide, allowing the DNA polymerases to continue extension of the new strand. 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. 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.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.

, Tth DNA polymerase) possess a reverse transcriptase activity, which can be activated by using manganese instead of magnesium as a cofactor (Myers and Gelfand, 1991). After this initial reverse transcription step to produce the cDNA template, basic PCR is carried out to amplify the target sequence. The efficiency of the first-strand synthesis reaction, which can be related to the RNA quality, also will significantly affect amplification results. At room temperature, PCR primers can anneal to template sequences that are not perfectly complementary. Since thermostable DNA polymerases have activity at these low temperatures (although in most cases the activity is less than 25%) the polymerase can extend misannealed primers. This newly synthesized region then acts as a template for primer extension and synthesis of undesired amplification products.The formation of nonspecific products and primer-dimer can compete for reagent availability with amplification of the desired product. Thus, hot-start PCR can improve the yield of specific PCR products. 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. Finally, the DNA polymerase can be maintained in an inactive state through chemical modification (Moretti, T. et al 1998). Activity is restored during initial denaturation, allowing hot-start PCR. 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.

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. Some qPCR technologies employ fluorescently labeled PCR primers instead of probes. 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. 1993; Bernard et al. 1998). 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.

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. One such approach includes a second primer pair that is specific for a “housekeeping” gene (i.e., a gene that has constant expression levels among the samples compared) in the reaction (Gaudette and Crain, 1991; Murphy et al. 1990). 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.g., different size, restriction digest pattern, etc.). 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, 1993; McCulloch et al. 1995), including the fact that many depend on final analysis by electrophoresis.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 free magnesium changes of 0.6mM observed in their experiments dramatically affected amplification yields in an allele-specific manner. They postulated that magnesium chloride precipitates as a result of multiple freeze-thaw cycles. Amplifications were performed using various Mg concentrations to demonstrate the effect on the amplification of a 1.8kb target luciferase gene. The reaction products were analyzed by agarose gel electrophoresis followed by ethidium bromide staining.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. The yellow dye migrates at a rate faster than primers ( 2 at a concentration of 7.5mM for a final concentration of 1.5mM. In most cases, this is an excess of enzyme, and adding more enzyme will not significantly increase product yield.Accurate dispensing of small volumes of enzyme solutions in 50% glycerol is difficult, so we strongly recommend preparing a reaction master mix, which requires a larger volume of each reagent, to reduce pipetting errors. 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.Reagents commonly used to purify nucleic acids (salts, guanidine, proteases, organic solvents and SDS) are potent inactivators of DNA polymerases. 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.

1993; Linquist et al. 1998). 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. For example, of a 4kb plasmid containing a 1kb target sequence, 25% of the input DNA is the target of interest. Thus, approximately 1,000,000-fold more human genomic DNA is required to maintain the same number of target copies per reaction. Common mistakes include using too much plasmid DNA, too much PCR product or too little genomic DNA as the template. Reactions with too little DNA template will have low yields, while reactions with too much DNA template can be plagued by nonspecific amplification. When reamplifying a PCR product, the concentration of the specific PCR product is often not known. We recommend diluting the previous amplification reaction 1:10 to 1:10,000 before reamplification. 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. The Tm also can be calculated using the Biomath Calculators. Numerous formulas exist to determine the theoretical Tm of nucleic acids (Baldino, Jr. et al.

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. 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. 1998). 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. 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, 2001; Kurata et al. 2001). 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. 2001). These probes also can be used to detect single nucleotide polymorphisms (Lee et al. 1993; Bernard et al. 1998). 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. GoTaq qPCR Systems contain BRYT Green Dye, which provides maximum amplification efficiency and greater fluorescence than SYBR Green. Many of these suggestions also apply when using other DNA polymerases. Template DNA concentration, chelating agents present in the sample (e.g., EDTA or citrate), dNTP concentration and the presence of proteins all can affect the amount of free magnesium in the reaction. In the absence of adequate free magnesium, Taq DNA polymerase is inactive. Excess free magnesium reduces enzyme fidelity (Eckert and Kunkel, 1990) and may increase the level of nonspecific amplification (Williams, 1989; Ellsworth et al. 1993). For these reasons, researchers should empirically determine the optimal magnesium concentration for each target. The effect of magnesium concentration and the optimal concentration range can vary with the particular DNA polymerase. 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.

1989; Rychlik et al. 1990). The formula below can be used to estimate the melting temperature for oligonucleotides: 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). 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. 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. 1993). 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, 1961). Concentrations of DMSO greater than 10% and formamide greater than 5% can inhibit Taq DNA polymerase and presumably other DNA polymerases as well (Varadaraj and Skinner, 1994). 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.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. 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. UNG treatment prevents replication of uracil-containing DNA by causing the DNA polymerase to stall at the resulting abasic sites. For UNG to be an effective safeguard against contamination, the products of previous amplifications must be synthesized in the presence of dUTP. This is easily accomplished by substituting dUTP for some or all of the dTTP in the reaction. Nonproofreading polymerases will readily incorporate dUTP into a PCR product, but proofreading polymerases incorporate dUTP much less efficiently (Slupphaug et al. 1993; Greagg et al. 1999; Lasken et al. 1996). 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, 1993), UNG treatment has the advantage that both single-stranded and double-stranded DNA templates will be rendered unamplifiable (Longo et al. 1990). Many of the important parameters discussed there also apply to RT-PCR. For a discussion of reverse transcriptases commonly used for RT-PCR, see the Thermostable Polymerases and Reverse Transcriptases section (below). Procedures for creating and maintaining a ribonuclease-free (RNase-free) environment to minimize RNA degradation are described in Blumberg, 1987. For optimal results, the RNA template, whether a total RNA preparation, an mRNA population or a synthesized RNA transcript, should be DNA-free to avoid amplification of contaminating DNA.

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. The specific 540bp amplicon is indicated.For example, a primer that anneals specifically to the 3?-end of the transcript (a sequence-specific primer or oligo(dT) primer) may be problematic when reverse transcribing the 5?-ends of long mRNAs or molecules that have significant secondary structure, which can cause the reverse transcriptase to stall during cDNA synthesis. 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. 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). We recommend using a final concentration of 1?M primer (50pmol in a 50?l reaction) as a starting point for optimization. More information on PCR primer design is provided in the PCR Primer Design section. 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. 1992; Chumakov, 1994).

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. All of the DNA polymerases mentioned below can be used to amplify first-strand cDNA produced by a reverse transcriptase, such as AMV RT, in two-enzyme RT-PCR. 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, 1990; Eckert and Kunkel, 1991). 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. 1992), where its activity is similar to that of Taq DNA polymerase.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-MLV reverse transcriptases. Recombinant Tth DNA polymerase has been shown to exhibit RNase H-like activity (Auer et al. 1995).

Pfu DNA polymerase can be used alone to amplify DNA fragments up to 5kb by increasing the extension time to 2 minutes per kilobase. It is also used in blends with DNA polymerases lacking the proofreading function, such as Taq DNA polymerase, to achieve longer amplification products than with Pfu DNA polymerase alone (Barnes, 1994). However, the proofreading activity can shorten PCR primers, leading to decreased yield and increased nonspecific amplification. Reverse transcriptases use an RNA template to synthesize DNA and require a primer for synthesis, like other DNA polymerases. Polymerization from a primer then proceeds as for DNA-dependent DNA polymerases.For these templates, M-MLV RT or M-MLV RT, RNase H minus, may be a better choice. The enzyme also has DNA-dependent DNA polymerase activity at high enzyme levels (Roth et al. 1985). M-MLV RT is used in a variety of applications, including cDNA synthesis, RT-PCR and RACE (Gerard, 1983). However, for short templates with complex secondary structure, AMV RT or M-MLV RT, RNase H minus, may be a better choice due to their higher optimal temperatures. M-MLV RT is less processive than AMV RT, so more units of M-MLV RT may be required to generate the same amount of cDNA (Schaefer, 1995). 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). Combine the following: Centrifuge 10 seconds in a microcentrifuge.