Abstract
The replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNA polymerases were compared using a PCR-based forward mutation assay. Average error rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3 × 10 −6 ) < Deep Vent (2.7 × 10 −6 ) < Vent (2.8 × 10 −6 ) < Taq (8.0 × 10 −6 ) ≪ exo −Pfu and UlTma (∼5 × 10 −5 ). Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2–3 mM MgSO 4 and 100–300 µM each dNTP and at pH 8.5–9.1. Under these conditions, the error rate of exo −Pfu was ∼40-fold higher (5 × 10 −5 ) than the error rate of Pfu . As the reaction pH was raised from pH 8 to 9, the error rate of Pfu decreased ∼2-fold, while the error rate of exo −Pfu increased ∼9-fold. An increase in error rate with pH has also been noted for the exonuclease-deficient DNA polymerases Taq and exo − Klenow, suggesting that the parameters which influence replication error rates may be similar in pol I- and α-like polymerases. Finally, the fidelity of ‘long PCR’ DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNA polymerase mixture and a Klen taq/Pfu DNA polymerase mixture were found to be less than the error rate of Taq DNA polymerase, but ∼3–4-fold higher than the error rate of Pfu DNA polymerase.
Introduction
The use of high fidelity DNA polymerases in the polymerase chain reaction (PCR) is essential for reducing the introduction of amplification errors in PCR products that will be cloned, sequenced and expressed. Several thermostable DNA polymerases with 3′→5′ exonuclease-dependent proofreading activity ( Pfu, Vent, Deep Vent and UlTma ) have been introduced for high fidelity PCR amplification ( 1–3 ). Flaman et al. have reported that the error rate of Pfu was 5- and 30-fold lower than the error rates of the proofreading enzymes Deep Vent and UlTma , respectively ( 4 ). Using several different fidelity assays, the error rate of Pfu has been found to be ∼10-fold lower than that of the non-proofreading enzyme Taq ( 1 , 4 , 5 ).
The parameters which contribute to the replication fidelity of DNA polymerases need to be investigated, as very little is known about the molecular features of these enzymes which give rise to variations in replication fidelity and mutational spectra. A number of factors are thought to contribute to the overall fidelity of a DNA polymerase (reviewed in 6–8 ). These parameters include the tendency of a polymerase to incorporate incorrect nucleotides and the presence of an integral 3′→5′ exonuclease activity which can remove mispaired bases (proofreading activity).
The importance of proofreading activity to replication fidelity has been demonstrated for both the Klenow fragment ( 9 ) and for Vent polymerase ( 10 ), which exhibit 10- and 5-fold increases in error rates, respectively, when the associated 3′→5′ exonuclease activity is inactivated. The contribution of proofreading activity to DNA polymerase fidelity is also evident when the error rates of proofreading and non-proofreading enzymes are compared. Kunkel has noted that the average base substitution error rates exhibited by non-proofreading DNA polymerases range from 10 −2 to ≥ 10 −6 , while the error rates of proofreading enzymes range from 10 −6 to 10 −7 ( 7 ). The parameters which contribute to error rate variations among proofreading enzymes may reflect inherent differences in 3′→5′ exonuclease activity, the tendency to discriminate mispafred versus correctly paired bases and/or the efficiency of shuttling between polymerization and proofreading modes.
Recently, mixtures of non-proofreading and proofreading DNA polymerases have been reported to synthesize higher yields of PCR product and to allow amplification of longer templates than is possible with single enzyme formulations (‘long PCR’) ( 5 ). The addition of a low level of a proofreading enzyme (e.g. Pfu DNA polymerase) to PCR reaction mixtures has been proposed to improve the performance of non-proofreading polymerases (e.g. Taq DNA polymerase) by correcting mismatches introduced during PCR which prevent the efficient synthesis of full-length products ( 5 ). The PCR fidelity of DNA polymerase mixtures has not yet been determined, but error rates are likely to reflect the fidelity of the component polymerases and the ratio of non-proofreading to proofreading enzyme activities.
Pfu DNA polymerase has been found to be useful in high fidelity amplifications ( 1 , 4 ) of DNA targets up to 25 kb (K. Nielson, personal communication). In this report we use the previously described lacI PCR mutation assay ( 1 ) to compare the error rate of Pfu with an expanded number of PCR polymerases, including exo −Pfu, Deep Vent, Vent, UlTma and Taq , as well as ‘long PCR’ DNA polymerase mixtures. Polymerase error rates have been found to vary with buffer composition, including pH, Mg 2+ concentration and nucleotide concentration ( 11–13 ). PCR reaction conditions have been optimized with respect to fidelity for both Vent and Taq DNA polymerases ( 11 ). Buffer optimization studies with Pfu DNA polymerase were performed here to assess whether the fidelity of Pfu DNA polymerase could be further enhanced. Error rate comparisons between Pfu and exo −Pfu are expected to illuminate the contribution of proofreading activity to the fidelity of Pfu DNA polymerase. Finally, PCR fidelity comparisons between Pfu DNA polymerase and Pfu -containing DNA polymerase mixtures will allow evaluation of the contribution of the predominant non-proofreading enzyme to the error rate of ‘long PCR’ mixtures.
Materials and Methods
DNA polymerases
Cloned Pfu , exo −Pfu and Taq DNA polymerases were prepared at Stratagene. Deep Vent and Vent polymerases were purchased from New England BioLabs, UlTma was obtained from Perkin-Elmer and KlentaqLA (KTLA) was provided by Wayne Barnes (Washington University School of Medicine, St Louis, MO). Except where indicated, PCR amplifications were performed in the presence of buffers supplied by the manufacturers. The KTLA PCR buffer used was buffer PC2 of Barnes ( 5 ).
PCR fidelity assay
The fidelity of DNA replication during PCR was measured using a previously described assay ( 1 , 14 ). Briefly, a 1.9 kb sequence encoding lacIOZα was PCR amplified as described below with oligonucleotide primers containing 5′ Eco RI restriction sites ( 1 ). The amplified fragments were digested with Eco RI, purified by gel electrophoresis and ligated into λgt10 arms. The ligation reactions were packaged and the λ phage used to infect an a-complementing Escherichia coli host strain. Aliquots of infected cells were plated on LB plates with top agar containing either X-gal (1 mg/ml) or X-gal plus IPTG (1.5 mM). Error rates were calculated as described in the legend to Table 1 .
Average error rates of thermostable DNA polymerases during PCR a
PCR amplifications
Except where indicated, PCR amplifications were performed in 100 µl reaction volumes in the presence of the appropriate Tris-based buffer, using 5 U polymerase, 200 µM each dNTP, 250 ng each primer and 24 ng lacIOZ α target (50 ng lacIOZ α plasmid template). The PCR mixtures were denatured by heating at 95°C for 30 s. Thirty cycles of amplification were performed using the following conditions: 5 s at 95°C; 1 min at 55°C; 2.5 min at 72°C.
Results
PCR fidelity of thermostable DNA polymerases
Replication fidelities of thermostable DNA polymerases were compared using a previously described assay ( 1 ) which measures the frequency of mutations introduced into the lacI target gene during PCR amplification. PCR amplification was performed in the presence of each enzyme's optimal PCR buffer. All other PCR parameters remained constant, including the dNTP, primer and template concentrations, the PCR cycling parameters and the number of PCR cycles performed.
Pfu DNA polymerase exhibited the greatest PCR fidelity, with an average error rate of 1.3 × 10 −6 mutation frequency/bp/duplication ( Table 1 ). The lacI target size used in these calculations was estimated to be 349 bp, based upon the most recent analysis of lacI− mutant DNA sequences ( 15 ). Previous error rate calculations assumed a lacI target size of 182 bp ( 1 ). After recalculating error rates based on a lacI target size of 349 bp, the mean error rate of Pfu DNA polymerase obtained in this study (1.3 × 10 −6 mutation frequency/bp/duplication) was found to be similar to previous estimates obtained using an identical assay (0.8 × 10 −6 ; 1 ) or an alternative PCR-based assay employing a p53 target gene (≤ 1.0 × 10 −6 ; 4 ).
Average error rates of thermostable DNA polymerases were found to increase in the following order: Pfu (1.3 × 10 −6 ) < Deep Vent (2.7 × 10 −6 ) < Vent (2.8 × 10 −6 ) < Taq (8.0 × 10 −6 ) ≪ UlTma (5.5 × 10 −5 ). These results are in excellent agreement with the relative error rates measured by Flaman et al. ( 4 ), who reported that Pfu exhibits the greatest PCR fidelity, followed by Deep Vent, Taq and UlTma DNA polymerases. The relative error rates obtained here are also consistent with DGGE analyses showing that Pfu exhibits a lower error rate than Vent and Taq DNA polymerases ( 16 ). We found that relative error rates observed using the lacI screening assay were consistent from PCR reaction to PCR reaction.
The influence of template doublings ( d ) on error rate estimates of Pfu DNA polymerase was also examined ( Table 1 ). Amplification reactions described above and resulting in the Pfu error rate of 1.3 × 10 −6 employed 24 ng lacI target (10 10 copies). Approximately 10 doublings were observed in 30 PCR cycles. When the input lacI target DNA was decreased from 10 10 copies (24 ng) to 10 7 copies (0.02 ng), the number of template doublings increased from 9.7 (∼900-fold amplification) to 19.4 (∼700 000-fold amplification) after 30 cycles of PCR. The error rate of Pfu DNA polymerase varied from 0.7 to 1.3 × 10 −6 over the 1000-fold range of DNA target concentrations tested. Flaman et al. have also reported that polymerase error rates do not appear to be significantly influenced by the number of template doublings ( 4 ).
Optimization of the PCR fidelity of Pfu
We attempted to further improve the fidelity of Pfu by optimizing PCR reaction conditions. PCR error rates were measured at varying concentrations of MgSO 4 ( Fig. 1 ) and dNTPs ( Fig. 2 ) and at varying pHs ( Fig. 3 ). The indicated pH values are those measured at room temperature. Where noted, the pH of Tris buffers at elevated temperatures was estimated using the formula: pH T = pH 25°C + [( T °C − 25°C) × (−0.03 pH U/°C)] (where T is the reaction temperature; 17 ). The lowest error rates for Pfu were observed when PCR amplifications were performed in the presence of 2–3 mM MgSO 4 , 100–300 µM each dNTP and in a pH range between 8.5 and 9.1 (pH ∼7.1–7.7 at 72°C). These conditions have been found to give optimal yield of PCR product as well ( 18 ).
In the presence of 1 mM MgSO 4 and 800 µM total dNTPs, the error rate of Pfu was ∼3-fold higher than when PCR amplifications were performed in 2 mM MgSO 4 at the same dNTP concentration ( Fig. 1 ). The error rate did not vary significantly as the MgSO 4 concentration was increased from 2 to 10 mM (∼1.2–9.2 mM free Mg 2+ ). The error rate of Vent polymerase has also been shown to decrease significantly between 0.5 and 2 mM MgSO 4 in the presence of 2 mM total dNTPs and thereafter remains constant with increasing concentrations of free Mg 2+ ( 11 ). These results are in contrast to those reported for Taq, in which error rates are lowest at equimolar concentrations of MgCl 2 and dNTPs (1 mM) and increase with increasing concentration of free Mg 2+ ( 12 ). Error rate variations of Pfu and Vent likely reflect the Mg 2+ dependency of both proofreading and polymerase activities.
In Figure 2 , the error rate of Pfu was found to increase 2.4-fold as the total dNTP concentration was raised from 0.4 to 4 mM in the presence of a constant amount of free Mg 2+ (∼1.2 mM). These results are consistent with the observations of Clayton et al. ( 19 ), who report that high concentrations of dNTPs diminish the proofreading ability of exonuclease-proficient polymerases by increasing the efficiency of mispair extension. It is likely that the fidelity of Pfu DNA polymerase could be further increased by reducing the total dNTP concentration below 0.4 mM total dNTPs. However, using lower dNTP concentrations to increase the fidelity of PCR amplification reactions is not practical, as PCR product yields decrease significantly below 0.4 mM total dNTPs.
Variation of the PCR error rates of Pfu DNA polymerase with MgSO 4 concentration. PCR amplification was performed in buffer containing 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 0.1% Triton X-100, 100 µg/ml BSA, 200 µM each dNTP and varying concentrations of MgSO 4 (1–10 mM). Error rates shown are the average (± range) values obtained from two independent PCR amplifications.
Variation of the PCR error rates of Pfu DNA polymerase with dNTP concentration. PCR amplification was performed in buffer containing 20 mM Tris-HCl, pH 8.8, 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 0.1% Triton X-100, 100µg/ml BSA and varying concentrations of dNTPs (100–1000 µM each). The MgSO 4 concentration of the PCR mixtures was adjusted to give a constant free Mg 2+ concentration (1.2 mM) at each dNTP concentration: 1.6 mM MgSO 4 / 0.4 mM total dNTPs; 2 mM MgSO 4 /0.8 mM total dNTPs; 2.4 mM MgSO 4 / 1.2 mM total dNTPs; 3.2 mM MgSO 4 /2 mM total dNTPs; 5.2 mM MgSO 4 /4 mM total dNTPs. Error rates shown were normalized such that the mean mutation frequency for Pfu amplifications with 0.8 mM total dNTPs (assay internal control) was 1.3 × 10 −6 mutation frequency/bp/duplication. The average error rates (± range) from two independent PCR amplifications are shown.
In Figure 3 (inset), the error rate of Pfu was measured as a function of pH. The error rate of Pfu was found to decrease 4-fold between pH 7.5 and 8.5 in the presence of 2 mM MgSO 4 and 0.8 mM total dNTPs. Vent polymerase has also been reported to exhibit a significant decrease in error rate as the pH is increased from 7 to 8 in the presence of 2 mM MgSO 4 ( 11 ). For Taq DNA polymerase, a 2-fold increase in error rate was observed when the reaction pH was raised from 8 to 9 ( 11 ). Eckert and Kunkel have also reported that the base substitution and frameshift error rates of Taq ( 12 ) and exo − Klenow ( 13 ) increase >10-fold as the reaction pH is raised from ∼6.5 to 9.5 (25°C estimates of pH from 12 , 13 ).
Variation of the PCR error rates of Pfu and exo −Pfu DNA polymerases with pH. PCR amplification was performed in 20 mM Tris-HCl buffers whose pH values ranged from 7.5 to 9.1 (pH at 25°C). In addition, the buffer contained 2 mM MgSO 4 , 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 0.1% Triton X-100, 100 µg/ml BSA and 200 µM each dNTP. The average error rate of Pfu (open diamonds) is shown in comparison with exo −Pfu (filled diamonds) and in the accompanying inset. Error rates shown are the average (± range) values obtained from two independent PCR amplifications.
pH dependency of the fidelity of Pfu and exo −Pfu
The error rates of Pfu and exo −Pfu were compared to assess the contribution of 3′→5′ exonuclease activity to fidelity. In the presence of Pfu PCR buffer (2 mM MgSO 4 , 200 µM each dNTP, pH 8.8), exo −Pfu exhibited an error rate of 4.7 × 10 −5 mutation frequency/bp/duplication, which is ∼40-fold higher than that determined for exonuclease-proficient Pfu .
Figure 3 shows the error rate variation of exo −Pfu and Pfu as a function of pH. Exo −Pfu shows a dramatic increase in error rate (∼9-fold) as the reaction pH is raised from pH 8 to 9.1 (or from 6.6 to 7.7 at 72°C). In contrast to exo −Pfu , the error rate of Pfu decreased ∼2-fold in this pH range. Presumably, the fidelity of Pfu is maintained at high pH (pH 9) by enhanced proofreading activity, which accompanies the dramatic increase in nucleotide misincorporation occurring between pH 8 and 9.1 (identified using exo −Pfu ). These results and those reported by others for Taq and exo − Klenow ( 11–13 ) indicate that the error rates of exonuclease-deficient enzymes, Taq , exo − Klenow and exo −Pfu , are similarly increased by pH. The significance of the apparent biphasic relationship between error rate and pH is currently under investigation.
PCR fidelity of ‘long PCR’ DNA polymerase mixtures
The fidelities of Pfu and Taq DNA polymerases were compared with the fidelities of two Pfu -containing DNA polymerase mixtures ( Table 2 ). A Taq/Pfu (16 U:1 U) mixture was prepared and shown to amplify DNA targets >30 kb (data not shown). The Taq/Pfu mixture exhibited an average error rate of 5.6 × 10 −6 mutation frequency/bp/duplication when amplifications were performed in Taq PCR buffer. The mean error rate of the Taq/Pfu mixture was 30% lower than the mean error rate of Taq DNA polymerase when amplifications were conducted in Taq PCR buffer. When compared with the error rate of Pfu DNA polymerase in the same buffer system, the error rate of the Taq/Pfu mixture was found to be 6-fold higher.
Similar observations were made for a second ‘long PCR’ mixture, KTLA, which consists of Klentaq (N-terminally truncated Taq ) and Pfu DNA polymerases ( 5 ). When PCR amplifications were conducted as described in this report, KTLA exhibited a mean error rate of 3.9 × 10 −6 mutation frequency/bp/duplication, which was 3-fold higher than the error rate of Pfu DNA polymerase ( Table 2 ). When PCR conditions from Barnes ( 5 ) were used ( Table 2 , condition 2), KTLA exhibited a mean error rate (9.4 × 10 −6 ) which was 4-fold higher than the error rate of Pfu DNA polymerase assayed under identical conditions.
Error rate comparisons of DNA polymerases and ‘long PCR’ DNA polymerase mixtures
Discussion
The intrinsic properties of thermostable DNA polymerases which contribute to variation in PCR fidelity are not fully understood. In general, enzymes which possess an associated 3′→5′ exonuclease-dependent proofreading activity are thought to exhibit higher replication fidelity than non-proofreading DNA polymerases ( 7 ). Variation in fidelity among proofreading enzymes, such as Pfu, Vent and Deep Vent , may reflect differences in the rate of mispair excision, the level of discrimination between mispaired and correctly paired bases, the rate of mispair extension and/or the efficiency of shuttling the 3′ primer terminus between the polymerase and exonuclease active sites.
The contribution of 3′→5′ exonuclease activity to the PCR fidelity of Pfu was demonstrated directly by comparing the error rates of Pfu and exo −Pfu . The error rate of exo −Pfu was found to be 7-fold higher than the error rate of exo +Pfu at pH 8.0 and 40-fold higher at pH 8.8 ( Pfu PCR buffer).
Despite the importance of proofreading activity to the fidelity of Pfu and Vent ( 10 ), the presence of 3′→5′ exonuclease activity does not necessarily guarantee high fidelity DNA synthesis, as illustrated by UlTma DNA polymerase. The poor fidelity of UlTma DNA polymerase may be related to the relatively low level of 3′→5′ exonuclease activity exhibited by this enzyme. In a preliminary analysis of exonuclease activity, UlTma was found to exhibit significantly lower levels of 3′→5′ exonuclease activity than Pfu, Deep Vent and Vent DNA polymerases (A. Lovejoy, personal communication). However, other parameters are likely to contribute to low fidelity, since UlTma , an N-terminally deleted version of Thermatoga maritima DNA polymerase ( 20 ), exhibits an ∼7-fold higher error rate than Taq , which is completely devoid of proofreading activity.
In the absence of proofreading activity, a DNA polymerase like Taq is thought to accomplish high fidelity DNA synthesis by inefficient incorporation of non-complementary dNTPs and a reduced tendency to extend from mismatched 3′ primer termini. Huang et al. ( 21 ) have shown that, with the exception of C-T mispairs, Taq polymerase exhibits ∼100–1000-fold greater discrimination against mispair extension, as compared with avian myeloblastosis and HIV-1 reverse transcriptases, which extend most mispairs permissively. The rate at which DNA polymerases extend from mispaired 3′ primer termini, however, does not contribute to the actual fidelity of non-proofreading enzymes. The mismatch extension rate only contributes to fidelity in the sense that if the mismatch is extended inefficiently, the DNA will not be replicated to completion and the mutation will not be scored. Therefore, the mispair extension rate influences the number of detected mutants, rather than reflecting the inherent fidelity of a non-proofreading DNA polymerase.
The observed 6-fold difference in error rate between Taq (8 × 10 −6 ) and exo −Pfu (4.7 × 10 −5 ) suggests that the misincorporation and/or misextension rates of Pfu (as measured with exo −Pfu ) are significantly higher than those of Taq . Apparently, a lower degree of discrimination against misinsertion or mispair extension errors can be tolerated when an associated proofreading activity is present, as is the case with exonuclease-proficient Pfu .
Further fidelity measurements with exo −Pfu revealed that the fidelity of dNTP incorporation was significantly influenced by the pH of the PCR buffer. The error rate increased by ∼9-fold as the pH was raised from pH 6.6 to 7.7 (pH at 72°C). The error rates of both Taq ( 12 ) and exo − Klenow ( 13 ) increase similarly at higher pH. Eckert and Kunkel have attributed the lower fidelity of exo − Klenow at high pH to an increase in both nucleotide misinsertion and mispair extension ( 13 ).
It is tempting to speculate that the lower fidelity of exo −Pfu at high pH may also reflect increased misinsertion and mispair extension, analogous to the observations made for exo − Klenow ( 13 ). If so, it would suggest that the parameters which contribute to fidelity are similar, despite the structural differences which are thought to exist between the α-like (exo −Pfu ; 22 ) and pol I-like (exo − Klenow and Taq ) DNA polymerases. For example, the observed variation in error rates with pH suggests that an active site histidine residue may play a role in fidelity, possibly in the discrimination of mismatched 3′ primer termini. Alternatively, protonation of the primer, template or substrate dNTP may enhance error discrimination ( 13 ). Finally, pol I- and α-like polymerases may undergo a similar conformational change at low pH which may alter template binding properties, thereby improving error discrimination. Such a mechanism was proposed for exo − Klenow by Eckert and Kunkel ( 13 ) and was supported by additional data showing that lower error rates at low pH were accompanied by an increase in polymerase processivity.
The relative error rates for Pfu, Vent and Taq were found to parallel the terminal transferase activities of DNA polymerases. Hu ( 23 ) has compared the tendency of DNA polymerases to catalyze the addition of non-template-directed bases to the 3′-end of a DNA fragment (terminal transferase activity). Terminal transferase activity is high in Taq but low (Klenow and Vent ) or absent ( Pfu, T4 and T7 ) in proofreading enzymes, which presumably edit the misextended base. The absence of terminal transferase activity appears to correlate with high fidelity. Fidelity measurements compiled by Cha and Thilly show that the error rates of Pfu, T4 and T7 DNA polymerases are lower than the error rates of Vent and Klenow ( 16 ). Thus, the parameters which give rise to terminal transferase activity may be similar to those which contribute to lower fidelity. The lower error rate and lack of terminal transferase activity for Pfu (as compared with Vent ) may be the result of a reduced tendency of Pfu to incorporate a mismatch or a base opposite an abasic site. Alternatively, Pfu may excise misincorporated bases more readily or shuttle between the exonuclease and polymerase active sites more efficiently.
Finally, fidelity comparisons with Pfu -containing ‘long PCR’ DNA polymerase mixtures have shown that the error rate of mixtures appears to be intermediate between the error rate of Pfu and the non-proofreading DNA polymerase. The lower error rate of a Taq/Pfu mixture, as compared with Taq alone, suggests that Pfu is editing a certain percentage of mismatches that have been introduced by Taq during the PCR process. Editing may occur at the 3′-terminus after Taq has introduced a mismatch and dissociated from the incomplete PCR product ( 5 ). In the absence of Pfu, Taq presumably extends some of these putative stalling mismatches during the course of the PCR process; otherwise the mutations would not be scored in the lacI − screening assay and there would be no apparent difference in error rate between Taq and the Taq/Pfu mixture. Pfu may also reduce the overall error rate of Taq DNA polymerase by degrading Taq -generated duplex DNA containing mismatches and resynthesizing the correct sequence.
Although the error rate of the Taq/Pfu mixture is somewhat lower than the error rate of Taq alone, it is still 4–6-fold higher than the error rate of Pfu alone ( Table 2 ). These results indicate that the majority of PCR products are synthesized by Taq . This result is not surprising, since Taq is present in this particular mixture at a 16-fold higher polymerase unit concentration than Pfu DNA polymerase. Hence, the misincorporation rate of Taq DNA polymerase contributes significantly to the error rate of Taq/Pfu DNA polymerase mixtures.
KTLA, a ‘long PCR’ mixture of Klentaq and Pfu DNA polymerases, was also found to exhibit an error rate significantly higher than the error rate of Pfu . Our results aie inconsistent with the results of Barnes ( 5 ), who has compared the error rates of Pfu , Klentaq and KTLA-64 (∼640 U Klentaq:1 U Pfu ) using a similar PCR forward mutation assay based on the mutational target gene lacZ . Barnes reported that the error rate of the KTLA mixture was 2-fold lower than the error rate of Pfu DNA polymerase ( 5 ). There are several differences between the Barnes assay and the assay performed here, including PCR amplification conditions (see Table 2 legend), number of clones screened [500–4200 clones/1 PCR in Barnes ( 5 ) versus 10 000–50 000 clones/PCR/4 PCRs in this study] and the mutational target gene used ( lacZ versus lacI ), as well as possible unknown variations in the KTLA mixtures. The results in Table 2 demonstrate that differences in the PCR amplification conditions employed are not likely to contribute to the differences in relative error rates observed in the two studies. Fidelity analyses of additional DNA polymerase mixtures are currently under way to help elucidate the role of component enzymes and buffer composition in the fidelity of ‘long PCR’ amplifications.
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