Traditional hot start additives unnecessary
Kirk B. Nielson Janice Cline Holly Hogrefe
Stratagene Cloning Systems, Inc.
This article describes the important, novel application of Stratagenes Pfu DNA polymerase for minimizing PCR cold start mismatched primer-extension reactions. These mismatched extension reactions result in the synthesis of undesirable artifactual PCR products. The problem of mismatched extension reactions may be alleviated by using Taq DNA polymerase in conjunction with hot start techniques, which prevent nucleotide incorporation prior to the first PCR denaturation step. Pfu DNA polymerase,, * which exhibits minimal activity at problematic lower temperatures, obviates the need for Taq DNA polymerase-based hot start procedures.
Pfu DNA polymerase has the lowest error rate of any thermostable DNA polymerase studied to date.1,2 This feature makes it the preferred polymerase for techniques that require high-fidelity DNA synthesis by the polymerase chain reaction (PCR). Applications for Pfu DNA polymerase include gene cloning and expression, sequencing and site-directed mutagenesis. Pfu DNA polymerase exhibits exceptionally high thermostability3 and requires a relatively high temperature for optimal activity (Topt75C), characteristics that have significant implications in PCR applications. Most notable is Pfu DNA polymerases relatively low activity, in comparison to other commonly used PCR enzymes, at temperatures ranging from 22 to 50C. Minimal polymerase activity at these temperatures should result in fewer mispaired primer-extension reactions than occur with Taq DNA polymerase, which exhibits significantly higher activity at temperatures used in the primer-annealing phase of PCR (~50C).
The relatively high thermostability and Topt < /sub>of Pfu DNA polymerase are not unusual when one considers the natural habitat from which it was derived. Pfu DNA polymerase was cloned from the hyperthermophilic archaeon Pyrococcus furiosus, which grows optimally at 100C in geothermal marine sediments.4 In contrast, Taq DNA polymerase was cloned from the thermophilic eubacterium Thermus aquaticus, which grows optimally at 70C in thermal springs.4
In order to demonstrate the difference in activity vs. temperature profiles between Taq and Pfu DNA polymerases, the polymerase activity of the two enzymes was compared over a range of temperatures encountered in PCR amplifications. The activities of cloned Taq and cloned Pfu DNA polymerases were compared at equal unit concentrations at temperatures ranging from 15 to 100C. The percent of maximal polymerase activity is plotted as a function of temperature in figure 1. Taq DNA polymerase exhibited optimal (84%) polymerase activity between 60 and 74C and 27% to 70% activity between 40 and 50C. Pfu DNA polymerase, on the other hand, exhibited optimal activity between 70 and 77C and only 2% to 8% activity between 40 and 50C. No significant differences in activity vs. temperature profiles were observed between cloned and native DNA polymerases (data not shown). Polymerization by Pfu DNA polymerase decreased dramatically at temperatures above 75 to 80C, presumably because the primer-template duplex dissociates at elevated temperatures. The Tm of the 40-base primer used in the primer-extension assay was calculated to be approximately 92.6C.5
Pfu DNA polymerases low activity at or below 50C may represent one of its most important advantages in PCR. The use of Pfu DNA polymerase should lead to fewer mispaired primer-extension reactions than occur with Taq DNA polymerase. Mismatched primer-extension reactions are most likely to occur during (1) preparation of the PCR reaction mixtures, (2) ramping to the initial denaturation temperature and (3) successive primer-annealing steps (typically performed at 45 to 55C). Undesirable PCR products, which are typically shorter than the desired target, may be preferentially amplified when they become established, subsequently interfering with amplification, detection and quantification of the desired target. Since Taq DNA polymerase exhibits significantly higher polymerase activity than Pfu DNA polymerase at lower temperatures (figure 2), Taq DNA polymerase has a greater potential for extending mispaired primers that anneal during these three low-temperature phases of PCR amplification.
Hot start PCR protocols have been developed to reduce or prevent mispaired primer-extension reactions from occurring prior to the initial denaturation step in Taq DNA polymerase-based PCR amplifications.6,7 In these protocols, a critical PCR component (polymerase, magnesium or dNTPs) is manually withheld by separation from the reaction by a wax barrier,8 or the polymerase is reversibly inactivated by binding to an antibody, oligonucleotide or protein inhi bitor9 until a stringent primer-annealing temperature has been attained (usually 60 to 95C).
Since hot start improvements are most noticeable when amplifying complex, low copy number targets,6-8 we performed PCR comparisons using several genomic DNA targets that tend to generate troublesome cold start artifacts. The performance of Pfu DNA polymerase was compared to Taq DNA polymerase alone, Taq DNA polymerase in conjunction with various hot start techniques or AmpliTaq Gold DNA polymerase, which is a heat-activated version of AmpliTaq enzyme. The results from amplifying a 2-kb target from a single copy region of a transgenic mouse genome are shown (figure 2A). PCR amplifications used equivalent polymerase units of Taq DNA polymerase (alone or in conjunction with a Taq DNA polymerase-specific antibody or magnesium wax beads), AmpliTaq Gold DNA polymerase or Pfu DNA polymerase. Similar comparisons were performed using a 120-bp target in the human fucosidase gene (figure 2B) and a 230-bp target from the Epstein-Barr virus genome (figure 2C).
The PCR results shown in figure 2A, 2B and 2C demonstrate that specificity and yield of the desired amplification target are improved to varying extents when hot start techniques are used with Taq DNA polymerase (in figure 2A, 2B and 2C, compare lanes 3-8 to lanes 1, 2). For the 2-kb target (figure 2A), undesired PCR products were eliminated when Taq DNA polymerase amplifications were performed in conjunction with Taq DNA polymerase-specific antibody or magnesium wax beads. The 2-kb target, however, could not be amp lified with AmpliTaq Gold DNA polymerase under the conditions used. For the 120-bp target (figure 2B), extraneous products were synthesized with Taq DNA polymerase but not with AmpliTaq Gold DNA polymerase. The addition of Taq DNA polymerase-specific antibody or the magnesium wax beads did not eliminate the synthesis of artifacts. Finally, with the 230-bp target (figure 2C), undesired PCR products were absent when amplifications were conducted with Taq2000 DNA polymerase in the presence of magnesium wax beads or with AmpliTaq Gold DNA polymerase; however, PCR product yield was significantly reduced in reactions using AmpliTaq Gold DNA polymerase (lanes 5-6). For the 230-bp target, the addition of Taq DNA polymerase-specific antibody did not reduce the synthesis of undesired products by Taq DNA polymerase.
In total, our results demonstrate the variability of hot start techniques in eliminating the synthesis of undesired products by Taq DNA polymerase. In contrast, Pfu DNA polymerase (in figure 2A, 2B and 2C, lanes 9 and 10) consistently generated only the desired PCR product, and yields of PCR products were comparable to those generated with the best Taq DNA polymerase plus hot start combinations (figure 2A, lanes 3, 4, 7 and 8; figure 2B, lanes 5, 6; figure 2C, lanes 7, 8). The relatively poor performance of hot start techniques for amplifying the 120-bp and 230-bp targets suggests that mispaired primer-extension reactions may be occurring at primer-annealing steps that take place after the first PCR cycle. Of course, current hot start techniques are ineffective after the initial denaturation step. The use of Pfu DNA polymerase, which exhibits minimal polymerase activity at lower, nonstringent primer-annealing temperatures, is an effective solution for reducing the mispaired primer-extension reactions that occur in later PCR cycles.
In addition to exhibiting a relatively high Topt, Pfu DNA polymerase shows remarkable thermostability when compared to other thermophilic DNA polymerases.3 For example, Pfu DNA polymerase has a half-life of 18 to 25 hours at 95oC, while Taq DNA polymerase has a half-life of less than 1 hour (data not shown). The extreme thermostability of Pfu DNA polymerase allows higher denaturation temperatures to be used, which may increase the yield and purity of the desired PCR product. Specific research examples include the successful amplification of GC-rich PCR targets in the following human genes: dopamine D4 receptor gene (D4DR),10 proopiomelanocortin gene (POMC),10 and fragile X site in Xq27.3.11 These successful PCR amplifications were accomplished using Pfu DNA polymerase and a denaturation temperature of 98oC.
The following properties of Pfu DNA polymerase make it ideally suited for many PCR applications: (1) the lowest error rate of any thermostable DNA polymerase analyzed to date,1-2 which is an advantage in studies that require cloning, sequencing, gene expression and site-directed mutagenesis; (2) can be used alone to amplify relatively long PCR targets (up to 12 to 25 kb) if given adequate extension time,12 which is important if long gene sequences or vector constructs must be correctly amplified for further sequence analysis; (3) unusually high thermostability allows Pfu DNA polymerase to successfully amplify GC-rich PCR targets without the addition of adjuncts (such as D MSO) that can be potentially damaging to the DNA template or to polymerase fidelity10,11 and (4) minimal polymerase activity at temperatures at or below 50oC, which may contribute to fewer undesirable PCR artifacts. Pfu DNA polymerase provides an alternative to Taq DNA polymerase, alone or in conjunction with a hot start technique, for increasing the yield and specificity of PCR products.
The DNA polymerase activity of Taq and Pfu DNA polymerases was measured by monitoring the incorporation of [3H]TTP into high-molecular-weight DNA using primed M13mp18(+) single-stranded template. The 40-base primer (54% GC) anneals to the multiple cloning region. Primer-extension reactions were carried out in each enzymes recommended PCR buffer in the presence of 200 M dATP, dCTP and dGTP, 200 M TTP (including 10 M [3H]TTP; specific activity of 20.9 Ci/mmole) and 1 g primer-annealed M13 DNA per 100-l reaction. Reactions were preincubated at the indicated temperatures, and polymerization was initiated with the addition of cloned Taq or cloned Pfu DNA polymerase to a final concentration of 10 U per 200-l reaction. (Unit concentrations of Taq and Pfu DNA polymerases were determined in the same assay by measuring nucleotide incorporation into activated calf thymus DNA at 72C.) After 10 minutes, polymerization was stopped by transferring the reactions to ice. From each reaction, 5 l was transferred to a chilled DE81 DNA-binding filter. Unincorporated [3H]TTP was removed by washing the filters in 8 changes of 2X SSC. Filters were added to scintillation cocktail and incorporated radioactivity was measured. Previous assays had demonstrated that the polymerase concentrations and extension time used for these reactions gave incorporation values that fell within the linear range of the assay for each reaction temperature. The mean % activity ( + one stand ard deviation) is shown for two independent assays, each performed in duplicate.
All PCR amplifications were performed under identical conditions, using 2.5 U of Taq2000 DNA polymerase, Taq2000 DNA polymerase plus a hot start technique, AmpliTaq Gold DNA polymerase or cloned Pfu DNA polymerase. The following reaction components were added to each Stratagene 600-l thin-wall tube: 250 ng of DNA template, 250 ng of each oligonucleotide primer and 1X of the appropriate reaction buffer in a 100-l ml reaction volume. Taq, cloned Pfu or GeneAmp PCR buffers were used for Taq2000, cloned Pfu or AmpliTaq Gold DNA polymerase amplifications, respectively. Cycling parameters are described in each figure legend. The amplified reaction products were electrophoresed on a vertical 6% acrylamide, 1X TBE gel for 2 hours at 50 V, stained with ethidium bromide and imaged with the Eagle Eye II still video system.
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