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1. Stratagene using thin walled PCR tubes and 7 2 ng target 15 ng lacIOZa plasmid Sixteen cycles of amplification were performed using the following conditions 30 s at 99 C 30 s at 67 C 3 min at 68 C Defined in the legend to Table 1 The Tag Pfu mixture consists of Tag 5 U ul and Pfu 0 31 U ul DNA polymerases d indicates range of duplicate measurements 3550 Nucleic Acids Research 1996 Vol 24 No 18 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 proofreading2. 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 o 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 a 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 Tag 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
3. mixture was 30 lower than the mean error rate of Tag DNA polymerase when amplifications were conducted in Jag 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 Klentagq N terminally trun cated Taq and Pfu DNA polymerases 5 When PCR amplifica tions were conducted as described in this report KTLA exhibited a mean error rate of 3 9 x 10 mutation frequency bp duplica tion 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 x 10 which was 4 fold higher than the error rate of Pfu DNA polymerase assayed under identical conditions Table 2 Error rate comparisons of DNA polymerases and long PCR DNA polymerase mixtures PCR condition DNA polymerase No of PCRs 1 Pfu 10 Taq 11 Taq Pfu 16 1 Taq buffer 8 Pfu buffer 11 KTLA 2 2 Pfu 2 KTLA 2 Template doublings gt Error rate x 10 SD 8 7 9 7 1 30 2 8 7 11 8 0 3 9 9 7 10 5 6 1 6 9 7 11 7 6 1 2 9 7 3 9 0 14 8 1 2 3 0 24 8 1 9 4 0 94 aPCR condition 1 is described in Materials and Methods PCR amplification PCR condition 2 is from Barnes 5 and differs in the following respects PCR amplifica tions were performed on a Robocycler 40
4. polymerase has also been reported to 240 230 220 exo Piu Error rate x10 pH Figure 3 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 MgSOQ 10 mM KCI 10 mM NHy4 2SOzq 0 1 Triton X 100 100 ug ml BSA and 200 UM 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 exhibit a significant decrease in error rate as the pH is increased from 7 to 8 in the presence of 2 mM MgSO 11 For Tag 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 gt 10 fold as the reaction pH is raised from 6 5 to 9 5 25 C estimates of pH from 12 13 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 gt 5 exonuclease activity to fidelity In the presence of Pfu PCR buffer 2 mM MgSQu 200 UM each dNTP Nucleic Acids Research 1996 Vol 24 No 18 3549 pH 8 8 exo Pfu exhibited an error
5. rate of 4 7 x 105 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 1s currently under investigation PCR fidelity of long PCR DNA polymerase mixtures The fidelities of Pfu and Tag 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 gt 30 kb data not shown The Taq Pfu mixture exhibited an average error rate of 5 6 x 10 mutation frequency bp duplication when amplifications were performed in Jag PCR buffer The mean error rate of the Tag Pfu
6. 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 Tag presumably extends some of these putative stalling mismatches during the course of the PCR process otherwise the mutations would not be scored in the Jac screening assay and there wo
7. 00 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 lach 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 REFERENCES 1 Lundberg K S Shoemaker D D Adams M W W Short J M Sorge J A and Mathur E J 1991 Gene 180 1 6 2 Kong H Kucera R B and Jack W E 1993 J Biol Chem 268 1965 1975 CON NN 16 17 18 19 20 21 22 23 Nucleic Acids Research 1996 Vol 24 No 18 3551 Abramson R D 1995 In Innis M A Gelfand D H and Sninsky J J eds PCR Strategies Academic Press San Diego CA Flaman J M Frebourg T Moreau V Charbonnier F Martin C Ishioka C Friend S H and Iggo R 1994 Nucleic Acids Res 22 3259 3260 Barnes W M 1994 Proc Natl Acad Sci USA 91 2216 2220 Echols H and Goodman M F 1991 Annu Rev Biochem 60 477 511 Kunkel T A 1992 J Biol Chem 267 18251 18254 Goodman M F Creighton S Bloom L B and Petruska J 1993 Crit Rev Biochem Mol Biol 28 83 1
8. 26 Kunkel T A 1988 Cell 53 837 840 Mattila P Korpela J Tenkanen T and Pitkanen K 1991 Nucleic Acids Res 19 4967 4973 Ling L L Keohavong P Dias C and Thilly W G 1991 PCR Methods Applicat 1 63 69 Eckert K A and Kunkel T A 1990 Nucleic Acids Res 18 3739 3744 Eckert K A and Kunkel T A 1993 J Biol Chem 268 13462 13471 Cline J Braman J and Kretz K 1995 Strategies Mol Biol 8 24 25 Provost G S Kretz P L Hamner R T Matthews C D Rogers B J Lundberg K S Dycaico M J and Short J M 1993 Mutat Res 288 133 149 Cha R S and Thilly W G 1995 In Dieffenbach C W and Dveksler G S eds PCR Primer Cold Spring Harbor Laboratory Press Plainview NY Good N E 1986 Biochemistry 5 467 476 Nielson K B Costa G L and Braman J 1996 Strategies Mol Biol 9 24 25 Clayton L K Goodman M F Branscomb E W and Galas D J 1979 J Biol Chem 254 1902 1912 Innis M A Myambo K B Gelfand D H and Brow M A D 1988 Proc Natl Acad Sci USA 85 9436 9440 Huang M M Arnheim N and Goodman M F 1992 Nucleic Acids Res 20 4567 4573 Mathur E J Adams M W W Callen W N and Cline J M 1991 Nucleic Acids Res 19 6952 Hu G 1993 DNA Cell Biol 12 763 770
9. 3546 3551 Nucleic Acids Research 1996 Vol 24 No 18 1996 Oxford University Press PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases Janice Cline Jeffery C Braman and Holly H Hogrefe Stratagene Cloning Systems 11011 North Torrey Pines Road La Jolla CA 92037 USA Received June 12 1996 Revised and Accepted July 29 1996 ABSTRACT The replication fidelities of Pfu Taq Vent Deep Vent and U Tma DNA polymerases were compared using a PCR based forward mutation assay Average error rates mutation frequency bp duplication increased as follows Pfu 1 3 x 10 6 lt Deep Vent 2 7 x 108 lt Vent 2 8 x 10 lt Tag 8 0 x 10 6 lt lt exo Pfu and UlTma 5 x 10 Buffer optimization experiments indicated that Pfu fidelity was highest in the presence of 2 3 mM MgSO and 100 300 uM each dNTP and at pH 8 5 9 1 Under these conditions the error rate of exo Pfu was 40 fold higher 5 x 10 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 repli cation error rates may be similar in pol l and o like polymerases Finally the fidelity of long PCR DNA polymerase mixtures was examined The error rates of a Taq
10. Pfu DNA polymerase mixture and a Klentaq Pfu DNA polymerase mixture were found to be less than the error rate of Tag 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 To whom correspondence should be addressed 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 integr
11. Vent 4 24 Vent 6 24 Taq 11 24 UlTma 2 24 Template doublings gt Nucleic Acids Research 1996 Vol 24 No 18 3547 PCR amplifications Except where indicated PCR amplifications were performed in 100 ul reaction volumes in the presence of the appropriate Tris based buffer using 5 U polymerase 200 UM each dNTP 250 ng each primer and 24 ng lacIOZq target 50 ng laclOZa 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 ac 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 x 10 mutation frequency bp duplication Table 1 The Jacl target size used in these calculations was estimated to be 349 bp based upon the most recent analysis of lac mutant DNA sequences 15 Previous error rate calculations assumed a lac target size of 182 b
12. activity to the fidelity of Pfu and Vent 10 the presence of 3 gt 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 U Tma 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 U Tma 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 Tag is thought to accomplish high fidelity DNA synthesis by inefficient incorporation of non complementary dNTPs and a reduced tendency to extend from mismatched 37 primer termini Huang et al 21 have shown that with the exception of C T mispairs Taq polymerase exhibits 100 1000 fold greater discri mination 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
13. al 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 to gt 10 while the error rates of proofreading enzymes range from 10 to 10 7 7 The parameters which contribute to error rate variations among proofreading enzymes may reflect inherent differences in 3 55 exonuclease activity the tendency to discriminate mispaired 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 po
14. es of Pfu DNA polymerase with dNTP concentration PCR amplification was performed in buffer containing 20 mM Tris HCl pH 8 8 10 mM KCI 10 mM NHy4 2SOq 0 1 Triton X 100 100ug ml BSA and varying concentrations of dNTPs 100 1000 uM each The MgSO concentration of the PCR mixtures was adjusted to give a constant free Mg2 concentration 1 2 mM at each dNTP concentration 1 6 mM MgSO 0 4 mM total dNTPs 2 mM MgSQO 0 8 mM total dNTPs 2 4 mM MgSOq 1 2 mM total dNTPs 3 2 mM MgSO 2 mM total dNTPs 5 2 mM MgSO 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 x 10 mutation frequency bp duplication The average error rates range from two independent PCR amplifications are shown 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 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 and 0 8 mM total dNTPs Vent
15. f 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 Tag DNA polymerases were prepared at Stratagene Deep Vent and Vent polymerases were purchased from New England BioLabs UlTma was obtained from Perkin Elmer and KlentagLA 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 lacIOZa was PCR amplified as described below with oligonucleotide primers containing 5 EcoRI restriction sites 1 The amplified fragments were digested with EcoRI purified by gel electrophoresis and ligated into AgtlO arms The ligation reactions were packaged and the 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 Table 1 Average error rates of thermostable DNA polymerases during PCR DNA polymerase No of PCRs Target ng Pfu 10 24 2 2 2 0 2 2 0 02 Deep
16. lymerases 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 acI 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 t concentration and nucleotide concentration 11 13 PCR reac tion conditions have been optimized with respect to fidelity for both Vent and Tag 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 containng DNA polymerase mixtures will allow evaluation o
17. mplate 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 3x 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 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 pHr pHa5 c T C 25 C x 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 MgSQg 100 300 UM 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 and 800 uM total dNTPs the error rate of Pfu was 3 fold higher than when PCR amplifica tions were performed in 2 mM MgSO at the same dNTP concentration Fig 1 The error rate did not vary significantly as the MgSO concentration was increa
18. 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 Tag 8x 10 and exo Pfu 4 7 x 10 suggests that the misincorporation and or misextension rates of Pfu as measured with exo Pfu are significantly higher than those of Tag 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 Jag 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
19. p 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 x 10 mutation frequency bp duplication was found to be similar to previous estimates obtained using an identical assay 0 8 x 10 1 or an alternative PCR based assay employing a p53 target gene lt 1 0 x 10 4 lacI plaques SD Error rated x10 SD 9 7 0 42 0 08 1 324 0 2 12 7 0 30 0 06 f 0 7 0 1 16 0 0 47 0 03 0 8 0 02 19 4 0 66 0 03 1 0 0 04 9 7 10 0 9 0 1 2 1 02 8 7 10 0 9 0 3 2 8 0 9 8 7 11 21 TL1 8 0 3 9 9 7 18 8 0 8 Oo ae 2 2PCRs were performed in each manufacturers recommended buffer all pH 8 8 in the presence of 200 UM each dNTP and 2 mM MgSO Pfu Deep Vent and Vent 2 mM MgCl U Tma or 1 5 mM MgCl Taq bTemplate doublings d were determined using the equation 2 amount of PCR product amount of starting target 24 ng ac target corresponds to 50 ng lacIOZa plasmid template The range of d obtained is indicated Mutant frequencies mf were determined by dividing the total number of blue plaques lac mutants on X gal plates by the total number of plaques containing a functional JacZa sequence blue plaques on X gal plus IPTG plates dError rates were calculated using the equation ER mf bp x d where mf is the mutation frequency bp is the number of detectable sites in Jacl 349 15 and d i
20. s the number of template doublings e indicates range of duplicate measurements fMutation frequencies for Pfu amplification of 0 02 2 ng target were normalized such that the mean mutation frequency for Pfu amplification of 24 ng target assay internal control was 0 42 3548 Nucleic Acids Research 1996 Vol 24 No 18 Average error rates of thermostable DNA polymerases were found to increase in the following order Pfu 1 3 x 10 lt Deep Vent 2 7 x 10 lt Vent 2 8 x 10 lt Tag 8 0 x 10 lt lt UlTma 5 5 x 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 Tag DNA polymerases 16 We found that relative error rates observed using the acI 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 Amplifica tion reactions described above and resulting in the Pfu error rate of 1 3 x 10 employed 24 ng Jacl target 10 9 copies Approximately 10 doublings were observed in 30 PCR cycles When the input lacI target DNA was decreased from 10 copies 24 ng to 10 copies 0 02 ng the number of te
21. sed from 2 to 10 mM 1 2 9 2 mM free Mg The error rate of Vent polymerase has also been shown to decrease significantly between 0 5 and 2 mM MgSO in the presence of 2 mM total dNTPs and thereafter remains constant with increasing concentrations of free Mg 11 These results are in contrast to those reported for Taq in which error rates are lowest at equimolar concentrations of MgCl and dNTPs 1 mM and increase with increasing concentration of free Mg 12 Error rate variations of Pfu and Vent likely reflect the Mg2 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 1 2 mM These results are consistent with the observations of Clayton et al 19 who report that high concentrations of dNTPs diminish the Error rate x10 mM MgSO Figure 1 Variation of the PCR error rates of Pfu DNA polymerase with MgSO concentration PCR amplification was performed in buffer containing 20 mM Tris HCl pH 8 8 10 mM KCl 10 mM NH4 2S04 0 1 Triton X 100 100 ug ml BSA 200 uM 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 3 6 2 m z 1 m 0 j 0 200 400 600 800 1000 uM each dNTP Figure 2 Variation of the PCR error rat
22. uld be no apparent difference in error rate between Taq and the Taq Pfu mixture Pfu may also reduce the overall error rate of Tag DNA polymerase by degrading Jag 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 Tag 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 are inconsistent with the results of Barnes 5 who has compared the error rates of Pfu Klentaq and KTLA 64 640 U Klentag 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 5
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