Building The Perfect Beast

Date: February 15, 1999Enhanced PCR Kits and Reagents Sigma's RedTaq DNA Polymerase facilitates gel loading. During the past decade, the polymerase chain reaction (PCR) has become one of the most versatile tools used in the molecular biology laboratory. Applications range from the analysis of genomic DNA samples and the production of DNA fragments for cloning systems to direct DNA sequencing. The reaction requires a minimum of reagents and equipment compared to many lab protocols and can be set up simply and quickly. It can be as useful to the solo underfunded bench scientist as it is to teams of production scientists using the most advanced automated equipment, generating significant amounts of data in a short time. The basic premise of the reaction is simple--DNA can be amplified many thousandfold or millionfold in a tube. A typical amplification reaction includes a minimal amount of the nucleic acid template, two oligonucleotide primers, deoxynucleotide triphosphates (dNTPs), reaction buffer, magnesium, optional extras, and a thermostable DNA polymerase. The components are mixed and put into an automated thermal cycler that cycles the reaction at different temperatures for varying amounts of time over and over, directing the reaction to denaturation, primer annealing, or elongation of the product. This product can be analyzed and quantified or used in other scientific endeavors. Utilization of DNA amplification has crossed over into disciplines outside of the common realm of molecular biology into botany, forensics, evolutionary science, and biotechnology. So if PCR is such a wonderfully simple, fast, and reliable technique that produces a mountain of results, what's the "catch"? What is it about PCR that can elate us or deflate us? The "catch" is that the reaction can be a beast to optimize, resulting in hours of frustration and futility in the lab. "PCR enhancement" can describe any number of manipulations of the reaction that optimize specificity and yield of the amplification. Enhancers may be new and expensive marketed reagents, or quite simply, tricks of the trade. Whatever its origin, an enhancement will become a necessity, and perhaps a welcome relief, in every PCR-oriented laboratory at some point. Choosing the appropriate enhancement option will depend on the intended application of the reaction, but one factor that falls into the "tricks of the trade" category and is paramount to every successful PCR of any type is the design of the primers. Typically, PCR primers are 15-30 bases in length, have a G+C base composition close to 50%, and are tailored to amplify a specific area of interest that is bracketed by the primer sequences. Occurrence of primer dimers can be minimized if the 3'-ends of the primers are not complementary. Avoiding three G or C bases in a row near the 3'-ends should reduce the likelihood of the formation of those pesky nonspecific amplification products. Both primers should have similar optimum annealing temperatures. The success of multiplex PCR, using multiple primer pairs in the same reaction to generate more than one product, can be dependent upon similar annealing temperatures of all the primers in the reaction. The primer pairs must amplify different regions of the molecule, both in individual pairs and in combinations of many primers, under a single set of reaction conditions; therefore, the more similar the annealing temperatures, the more likely that all target areas will be amplified. Primer pairs that work in individual pairs but not when combined may have improved activity after an ethanol precipitation in 0.3 M sodium acetate. Primer concentration is also a critical parameter in PCR, and even more so in multiplex PCR. If all the primers in a reaction anneal with the same amplification efficiencies, they may be used at the same concentrations. It is unlikely that this will always be the case, especially if one target area is more abundant than another; in this case, the concentration of the primer pairs targeting that area may need to be reduced. Finding the optimal concentration in any PCR will require some trial and error, but a good starting point is 1.0 µM of each primer. A series of titrated primer reactions, both increasing and decreasing concentrations, will confirm optimal primer concentration. Coupled reverse transcription and PCR amplification (RT-PCR) primer design and usage follow many of the same guidelines that apply to PCR in general. Primers should be designed from sequences in exons on opposite sides of an intron. A larger amplification product (which contains the intron) originating from contaminating genomic DNA will be distinguishable from the smaller desired mRNA product as separate bands on a gel. Improved amplification using the MasterAmp PCR Enhancer (with betaine). The apolipoprotein E gene was amplified with (Lane 2) or without (Lane 1) MasterAmp Enhancer. Image provided by Epicentre. Most polymerase chain reactions take place in a reaction buffer of 1.5 mM Mg, 10 mM Tris, and 50 mM KCl, at pH 8.3. Varying the pH of the buffer can be an enhancement option, although effects of a variant pH on other components of the reaction should be considered. Magnesium concentration can pose an interesting problem, especially in multiplex PCR. Free magnesium in the reaction is a driving force of Taq DNA polymerase activity, and a number of magnesium binders lurk in the background--inhibitory agents and chelators in the template, proteins, and dNTP. A standard 50-µl reaction requires a magnesium concentration in the range of 1.0-3.0 mM when using 1.0-1.5 units of Taq DNA polymerase. A multiplex reaction, which is actually multiple reactions taking place simultaneously, will most likely require increased concentrations of magnesium and polymerase, and possibly a slight increase in the dNTP concentration, for enhancement of specificity and product yield. Finding the optimal conditions that will ensure the potential amplification of all the desired products may mean weeks of toil in the lab, but it can be well worth the wait. A number of optional buffer extras that have been utilized in a variety of PCR protocols may be helpful in a disappointing reaction. Dimethyl sulfoxide (DMSO) has been a common additive in reactions in which the primers are less than ideal because of individual composition or disparity as a pair due to differences in melting temperature or size. DMSO acts to lower the melting temperature of the primers, which may be beneficial to the reaction, but it should be noted that in a high concentration (10%), DMSO inhibits Taq DNA polymerase activity. Addition of DMSO in the range of 0.9%-2.0% can enhance the quality of amplification by minimizing nonspecific binding, often a problem in multiplex PCR since the possibility of artifacts is increased with each additional primer. Betaine (N, N, N-trimethylglycine), a natural osmoprotectant, has been shown to alter the melting temperature of DNA such that GC-rich regions melt at temperatures more similar to AT-rich regions. It also appears that the addition of betaine improves the yield and specificity of PCR amplifications by relieving DNA polymerase pausing and expanding the optimal range for magnesium concentrations in the reactions. While the effects of betaine may be generally applicable regardless of the DNA polymerase used, the concentration of betaine required for amplification varies with the template/primer pair combination, so some optimization of betaine concentration may be required. Other additives that have been used to combat the problems of nonspecific binding or decreased product yield are low concentrations (0.01%-0.1%) of gelatin, glycerol, Tween-20, or Triton X-100. PCR optimizer kits are on the market that contain prepared buffers varying in pH, magnesium concentration, and buffer additives, thereby making the optimization process somewhat less painful and time-consuming. PCR is an extremely sensitive reaction that requires only nanogram amounts of target DNA for amplification to be successful, unlike many research protocols that require a stockpile of precious sample to complete any further analyses. The compilation of techniques for PCR-ready nucleic acid extractions ranges from down and dirty bacterial extractions that are nothing more than the lysis of the bacterial cell in sterile water to complex kits involving a number of purification steps and reagents. Many of the commonly used extraction reagents, especially guanidine, organic solvents, detergents, and salts, can inhibit the activity of the polymerase, although an ethanol precipitation of the sample at the end of the purification process will eliminate most of the inhibitors. Template concentration in the reaction varies according to the source and complexity of the nucleic acid. DNA isolated from a cloned plasmid, being present in higher numbers, will likely be used in lesser concentrations than genomic DNA isolated from cells. Recommended concentrations of plasmid samples range from 0.01 to 0.1 ng/reaction, whereas genomic sample concentrations should be from 10 to 500 ng/reaction. An overdose of template in the reaction can decrease specificity, as evidenced by extraneous bands or smearing of samples on a gel. Optimizing template concentration, much in the same manner as was described for the buffer components, may enhance the reaction. This may be necessary in a multiplex assay to ensure the equitable amplification of all desired targets, as templates of marginal quality may produce weaker signals for long bands than for short. Criteria for preparation and usage of RNA for RT-PCR differ little from those of DNA, although successful RT-PCR may be more dependent on the quality of the RNA. Isolation of the nucleic acid in the presence of a ribonuclease inhibitor will eliminate degradation due to RNase. Treatment of the sample with RNase-Free DNase will eliminate the aforementioned contaminating genomic DNA, thereby clarifying the true success or failure of the reaction. In the beginning there was Taq polymerase, and there is all that was. Today, selecting the appropriate enzyme for the specific PCR application has become a shopping extravaganza. Long or short, hot or cold, red or gold--what to choose? Taq polymerase is still the most widely used because of its relatively low cost and utility. But the contest is on to become the most popular enzyme, and "new and improved" could take the lead at any time. Amplitaq Gold™ DNA Polymerase is an inactive covalently modified form of Amplitaq™ DNA Polymerase designed to activate upon heating, resulting in enhanced specificity, lower background, and greater product yield. The required thermal induction of this enzyme makes reaction setup faster and easier than when using Amplitaq DNA Polymerase by eliminating the conventional hot-start step of heating the reaction prior to the addition of the enzyme. Instead, all components can be mixed together and then heated at 95šC for 10 minutes to initiate the reaction. Amplitaq Gold DNA Polymerase has been shown to enhance multiplex reactions, especially those with marginal primer balance or suboptimal conditions, by increasing the specificity of all desired targets. Promega's TaqBead™ Hot Start Polymerase utilizes a similar idea of temperature-controlled activation of the enzyme by encapsulating it in a wax bead that when heated to 60šC releases the polymerase into the reaction. Another new product designed to simplify PCR is REDtaq™ DNA Polymerase from Sigma, which is a blend of high-quality Taq polymerase with a dense inert red dye that acts as a tracer. It also serves as a tracking dye in gel samples, migrating a little faster than bromophenol blue. PCR was performed on five different primer-template sets using 5 units of Taq DNA polymerase alone or with the addition of 5 units of Taq Extender PCR additive. Image provided by Stratagene. Enzyme selection can be an important factor when dealing with difficult templates. Alternatives to Taq (Tbr, Tli, Pfu) can offer greater thermostability and/or fidelity. However, the high fidelity proofreading enzymes generally require careful optimization to give good yields. Enzyme mixes consisting of highly processive enzymes (Taq, Tbr) and a proofreading enzyme can provide improved fidelity and higher yields with long or difficult templates, and do so with less need for optimization. Amplification of long templates (>5 kb) is no longer as problematic as in the past with the development of a number of polymerases and polymerase systems that exhibit 3'->5' exonuclease proofreading activity, which is lacking in Taq DNA polymerase. DNAzyme EXT (MJ Research, Inc.), Expand Long Template PCR System, and Expand 20 kb PLUSPCR System (Roche Molecular Biochemicals) are enzyme "cocktails" made up of blends of thermostable polymerases that offer thermal stability, enhanced fidelity, proofreading ability, and the amplification of long (6-20 kb) and extra-long (20-40 kb) target sequences. RT-PCR requires an enzyme system that is capable of dual function: reverse transcription of the RNA into cDNA and PCR amplification of the cDNA. Promega's Access RT-PCR System and Roche Molecular Chemicals' Titan™ One Tube RT-PCR System combine the sensitivity of a multi-enzyme system with the convenience of a single-enzyme protocol. Both systems use avian myeloblastosis virus (AMV) for first-strand cDNA synthesis. Now that the beast has been assembled (ideally, in a dedicated area with dedicated tools) using near-perfect parts, decisions must be made about nurturing and ensuring growth to its full potential, based on time, temperature, and number of cycles. The denaturation step of the cycle varies little in most PCR applications with a temperature of 94š-96šC for a 30-second to two-minute interval. The annealing step is highly dependent on the melting temperature (Tm) of the oligonucleotides, which is calculated from the size and composition of each primer. Typically, the temperature of the annealing step ranges from 40šC to 60šC for 30 to 60 seconds. This step will likely require optimization, particularly in multiplex assays if the individual primers display significant differences in melting temperature. If the annealing temperature is too low, unwanted non-specific bands will appear on the gel, and if it is too high, expected bands may be missing. It is best to perform the reaction at several temperatures, starting at 5šC below any calculated Tm, to determine what is optimal. The extension step is usually carried out at 72šC, which is optimal for Taq polymerase, but the temperature can vary according to the enzyme system used. The extension time is determined by the length of the target sequence, but a general rule of thumb is to use a minimum of 1 minute/kb of amplicon. Once again, multiplex reactions may likely require some trial and error to enhance the amplification of fragments less robust than others. Generally, 20-30 cycles are sufficient for many applications. As primers and dNTPs are used up and the number of template molecules surpasses the supply of the polymerase, DNA synthesis becomes less efficient. To enhance product yield, a final extension of 5-15 minutes at 72šC allows the enzyme to finish any incomplete synthesis. Cycle parameters of RT-PCR are the same as those of standard PCR with the exception of the initial first-strand cDNA synthesis. This will employ a precycle 30 to 60 minute incubation at 37-48šC using AMV reverse transcriptase. Manipulation of cycle parameters during the amplification process may enhance the reaction and increase specificity. Touchdown or ramped PCR describes a cycling scheme in which the annealing temperature starts high and is lowered with successive cycles over a specified range of temperatures. For example, the annealing temperature in a 30-cycle profile starts out at 65šC and is lowered 1šC every second cycle to a "touchdown" temperature of 55šC at which it is maintained for the remaining 10 cycles. The higher annealing temperatures used in the beginning cycles of amplification will favor the specific priming of the longer target template, which may be present in small amounts, rather than shorter misprimed products. And because of the exponential nature of PCR, the amplification of the correct larger product will have a headstart over the production of spurious smaller bands, thereby increasing both target yield and specificity. The method of rapid cycle DNA amplification refers to the completion of 30 cycles in 10-30 minutes as opposed to the standard 2-4 hours necessary for standard amplification; time parameters are set in seconds rather than minutes. Not only is processing time decreased significantly, the production of nonspecific bands has also been shown to decrease with shorter cycles, probably because there is less time spent near the annealing temperature during the ramping transitions. The limitations of rapid cycling at this point are defined by available instrumentation-cyclic heating and cooling take time. But considering the importance and utility of PCR in the scientific kingdom, it is only a matter of time before the technology is perfected. And as the equipment becomes more advanced, the method becomes more simple. Is that not the perfect beast? Enhanced PCR Kits and Reagents The author, Debra Swanson, can be contacted at swans061@maroon.tc.umn.edu.

Building The Perfect Beast

Feb 14, 1999

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