With Regard to Fidelity: and Vent Polymerases Lucy L

Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Optimization of the Polymerase Chain Reaction with Regard to Fidelity: and Vent Polymerases Lucy L. Ling, Phouthone Keohavong, Cremilda Dias, and William G. Thilly Center for Environmental Health Sciences and Division of Toxicology, Whitaker College of Health Sciences and Technology, Massachusetts institute of Technology, Cambridge, Massachusetts 02139

The fidelity of DNA polymerases DNA amplification by the poly- Klenow fragment referred to in the used in the polymerase chain reac- merase chain reaction (PCR) O-3) can be original descriptions of DNA amplification (PCR) can be influenced by accomplished by DNA polymerases tion.(1,3,~~ However, the fidelity of Taq many factors in the reaction mix- from many sources.(1,2,4) Specific DNA has been reported to be 2 • 10 -4 er- ture. To maximize the fidelity of polymerases have been reported to ror/bp per duplication, (4,7-1~ which DNA polymerases in the PCR, pH, amplify with different efficiencies (i.e., renders it unsuitable for studies requir- concentrations of deoxynucleoside yield of sequence amplification per ing both low noise and high amplifica- triphosphates, and magnesium ion cycle) and fidelities (frequency of tion. Studies requiring a higher fidelity were varied. Denaturing gradient polymerase-induced errors), with the of amplification have used DNA poly- gel electrophoresis was used to kind and rate of error depending on merases such as modified T7 DNA separate the polymerase-induced the specific DNA polymerase and PCR polymerase (Sequenase) or T4 DNA mutants from wild-type DNA se- conditions. (4-9) Each error, once ini- polymerase,(4A3A4) which permitted quences. Thermolabile modified T7 tiated, is amplified along with the amplification with error rates of about DNA polymerase, therrnostable Taq, original wild-type sequences, increas- 3.4 x 10 -s and 0.3 x 10 -5 error/bp per and Vent DNA polymerases were ing the fraction of polymerase-induced duplication, respectively. (4) However, studied. Fidelity of all three DNA mutant sequences linearly with the both T4 and modified T7 DNA poly- polymerases was sensitive to con- number of amplification cycles. Under merases are thermolabile. centrations of deoxynucleoside low-fidelity conditions, this mutant Recently, a new thermostable DNA triphosphates, magnesium ion, and fraction can become significant: 40% polymerase called Vent DNA polymer- pH. Within conditions that permit- of the amplification products are ase was isolated and made commercial- ted efficient amplification, optimiza- mutant when a 200-bp fragment is ly available in a cloned form by New tion with regard to these three fac- amplified one million-fold by a DNA England Biolabs (Beverly, MA). Vent tors yielded an average error rate polymerase with an error rate of was reported to have a 3'-,5' in error/base pair incorporated of 10-4, (4) such as Klenow fragment.00) proofreading exonuclease. Os) Our 7.2 x 10 -s for Toq, 4.5 x 10 -s for For some purposes such as sequencing laboratory has been screening all avail- Vent, and 4.4 x 10 -s for modified T7 a wild-type gene, the polymerase- able thermostable polymerases and has (Sequenase) DNA polymerases. induced mutations are generally dis- found that this new thermostable DNA tributed over the sequence of interest polymerase, under conditions de- so that an accurate consensus sequence scribed for primer extension, permitted is usually obtained.O 1,12) For studies of amplification with a useful degree of fi- mutants within wild-type populations, delity. Reaction conditions such as pH, it is vital that the polymerase-induced concentrations of dNTP, and mag- mutant sequences do not mask the nesium ion can greatly affect the fidel- preexisting mutant sequences.Ol,13A4) ity of DNA polymerases.(S,7,9,16-20) Eck- Due to its thermostabiUty, Taq ert and Kunkel used the M13mp2 fidel- DNA polymerase has been the most ity assay and found that conditions of widely used DNA polymerase. It does" low pH with low and equimolar con- not have to be added every cycle, as centrations of dNTP to Mg 2+ increased was necessary for the thermolabile the fidelity of Taq.(7,9) We have ex-

1:63-699 byCold Spring Harbor Laboratory Press ISSN 1054-9803/91 $3.00 PCR Methods end Applications 63 Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

tended their observations using gel to ensure a homogeneous popula- PCR reaction mixture was removed denaturing gradient gel electrophoresis tion of the desired fragment of human and electrophoresed on 6% polyacryl- (DGGE) to separate and enumerate all HPRT exon 3 and flanking intron 2. amide gel together with a series of polymerase-induced mutant sequences This template was amplified from 10 s authentic standards whose numbers of from the correctly amplified sequences. copies to about 1012 copies and molecules are known. The gel was To maximize the fidelity of Taq, Vent, polyacrylamide gel purified. An aliquot stained with ethidium bromide and and Sequenase, varied concentrations (101~ copies) was further amplified the PCR products of the correct size of dNTPs, pH, and magnesium were lO0-fold, under identical conditions were measured by comparing the yield tested iteratively, and the optimal con- with primer P4 end-labeled by T4 poly- of the ultraviolet-induced fluorescence ditions with regard to these variables nucleotide kinase.(13) The number of emitted by the ethidium bromide have been defined. We have confirmed DNA copies generated was measured at molecules intercalated into the DNA of the finding of Eckert and Kunkel(7, 9) different cycles to permit calculation of the samples of DNA of the standard. and defined conditions in which the efficiency. Conditions that maintained The error of this method of quantita- Taq polymerase operates with a three- exponential increases in DNA per cycle tion is about twofold. However, this fold improvement in fidelity during were used throughout this work. The would affect the efficiency only slight- amplification in PCR relative to the 32p-end-labeled DNA of the correct size ly. For instance, a calculated efficiency most widely used reaction condi- was then purified from polyacrylamide of 70% due to 107-fold amplification tions. (4) More importantly, we find gel, boiled, and reannealed so that after 30 PCR cycles, would be 75% if that the thermostable Vent polymerase each strand of any PCR-generated the DNA was actually twice as much. under optimal conditions yielded a mutant homoduplexes could be hy- The efficiency of amplification was cal- PCR amplification with both high fi- bridized to the complementary strand culated from the formula: delity and high efficiency which of the excess wild-type products to Final DNA copy number should be of general use to researchers form heteroduplexes. These hetero- (1 + y)n = using PCR. duplexes were then separated from the Initial DNA copy number wild-type homoduplexes by denatur- MATERIALS AND METHODS ing gradient gel electrophoresis. For where Y is the efficiency per cycle and Materials each PCR condition, the total hetero- n is the number of PCR cycles under Taq polymerase was obtained from duplex and the correctly amplified conditions of exponential ampliflca- Perkin Elmer Cetus (Norwalk, CT). homoduplex fractions were measured tion.(1,2,14) Vent polymerase, purified from for the calculation of fidelity in er- Thermococcus litoralis, and its recom- ror/base pair incorporated per duplica- Denaturing Gradient Gel binant form cloned in Escherichia coli tion. Electrophoresis (DGGE) (designated Rec-Vent in this paper) To separate DNA duplexes containing were obtained from New England PCR Procedure only homologous wild-type sequences Biolabs (Beverly, MA). Sequenase pH, dNTP, and Mg 2§ concentrations (WT homoduplexes) from the mutant/ (modified T7 DNA polymerase) was ob- were experimental variables. MgSO 4 wild-type heteroduplexes, denaturing tained from United States Biochemicals was used for Taq and Vent, while gradient gel electrophoresis was (Cleveland, OH). 2'-deoxynucleoside MgC12 was used for Sequenase. For employed. (4) DNA was dissolved in a 5'-triphosphates (dNTPs) were ob- Vent and Taq, the PCR reaction mix- 30-~1 solution of 400 mM NaC1, 10 mM tained as 100 mM solution from ture was 10 mM KC1, 10 mM Tris buffer at pH 7.5, 2 mM EDTA, Pharmacia (Piscataway, NJ). Primer P1, (NH4)2SO 4, 0.1% Triton X-100, 0.1 boiled for 5 min, and reannealed for 5 5 ' -CATATATTAAATATACTCAC-3 '; ~g/~l BSA, 1 ~M each primer and 20 hr at 65~ The DNA was ethanol primer P2, 5'-TCCTGATITTA3TFC- mM Tris buffer. Four units of enzyme precipitated and separated on a TGTA-3 '; and primer P4, 5 ' -GACTGA- were added for every 100 ~l of reaction denaturing gradient gel. The gel con- ACGTCTTGCTCGAG-3' were obtain- volume, with 3 more units of Taq poly- sists of a 12.5% polyacrylamide gel ed from Synthetic Genetics (San Diego, merase added after 30 cycles. The (acrylamide/bis-acrylamide -- 37.5:1), CA). [y-32p]ATP (6000 Ci/mmole) was amplification cycle consisted of 94~ containing a linearly increasing gradi- from New England Nuclear (Boston, for 1 rain, 53~ for 2 min, and 70~ ent of denaturant, parallel to the direc- MA) and T4 polynucleotide kinase was for 2 min. For Sequenase, the reaction tion of electrophoresis, of 19-29% from New England Biolabs (Beverly, mixture was 3 pM each primer, and 10 (vol/vol) where 100% denaturant is 7 M MA). The reagents in the 5' DNA mM Tris buffer. The amplification cycle urea/40% formamide.(21) The gel was terminus labeling system were from consisted of boiling for 1 min, cooling run for 15 hr at 150 volts while sub- Bethesda Research Lab (Gaithersburg, down to 37~ (1 minute), adding 1 merged in TAE buffer (40 mM Tris/20 MD). unit (1 pl) of Sequenase, and incuba- mM acetic acid/2 mM EDTA, pH 8.3) at tion at 37~ for 2 min. (4) 60~ dried, autoradiographed, and re- Procedure corded on a phosphoImager screen The stock of DNA template for PCR Efficiency (Molecular Dynamics, Sunnyvale, CA). was obtained by amplification from To determine the efficiency of amp- human genomic DNA, followed by lification at the exponential phase of Determination of Fidelity purification with denaturing gradient DNA synthesis, a 5-~1 aliquot of the Fidelity of amplification was deter-

64 PCR Methods and Applications Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

mined by analyzing the denaturing shows the efficiencies and error rates gradient gel exposed to the phospho- using conditions commonly used in Imager screen. The amount of excita- PCR for Taq and Sequenase, (4) or in the tion from the radioactive bands in the case of Vent, those suggested by the heteroduplex region (from the origin supplier to be successful in primer ex- of the gel to the WT homoduplex tension. Table 1B shows the improve- band) and the band of WT homo- ment brought about by the optimiza- duplex were measured. The back- tion effort. ground noise, such as nonspecific DNA Figure 1 is an autoradiogram of a binding in the gel, was measured from denaturing gradient gel comparing the control lanes loaded with only DGGE polymerization-induced mutations by purified wild-type DNA homoduplex Sequenase, Taq, and Vent. The band of sequences that had been subjected to wild-type homoduplex indicated by an the identical treatment of boiling, arrow in the figure has been confirmed reannealing, and ethanol precipitation. by sequencing. The positions of the The background control samples were mutant/WT type heteroduplexes of run on the same denaturing gradient Vent are very similar to Taq, with a few gels as the experimental samples. The exceptions. As conditions were ad- heteroduplex fraction (HeF) due to justed for the improvement of fidelity, polymerase amplification was calcu- the positions of these heteroduplexes lated as: HeF = (Total heteroduplex remained the same with changes only counts - background heteroduplex in their intensity relative to the WT counts)/total counts. homoduplex. The specific Taq and Se- The fidelity was calculated (4) as: quenase point mutations correspond- ing to these bands have been previous- HeF ly reported. (4) f=-- bd Optimization began with the conditions of Table 1A and iteratively ex- where f is the error rate (errors/base amined the effects of dNTP concentra- pair incorporated.duplication); HeF is tion, pH, and then magnesium ion FIGURE 1 Analysis by DGGE of the PCR heteroduplex fraction; b is the length concentration. The effects of dNTP products after 109-fold amplification by Se- of the single-strand low melting concentration on fidelity are shown in quenase, Taq, and Vent DNA polymerases. domain in which mutants can be Figure 2. In a first round of experi- The arrow points to the position where cor- detected (104 bp); and d is the number ments, the dNTP concentrations that rectly amplified wild-type homoduplex of DNA duplications (30). permitted each polymerase efficiency focus. to exceed 35% were defined. Within RESULTS this range, Taq displayed some im- Table 1 shows the values of pH, dNTPs, provement in fidelity by either increas- shown on Figure 2, efficiencies were and magnesium with their correspond- ing or decreasing dNTP concentrations. invariant for all polymerases, -90% for ing efficiencies and fidelities that were The fidelity of Vent was somewhat im- Sequenase, -70% for Taq and Vent. observed in the studies with Se- proved by increasing dNTP as was that Based on these observations, 0.05 mM, quenase, Taq, and Vent. Table 1A of Sequenase. Within the ranges 0.5 mM, and 3.5 mM dNTP for Taq, Vent, and Sequenase, respectively, were used in the next stage, study of pH effects. TABLE 1 Summary of the PCR Conditions The efficiencies of Vent and Se- quenase were relatively invariant in Error rate the pH range of 7-9, but the efficiency DNA dNTP Mg Efficiency (error/bp of Taq decreased markedly below pH 8. polymerase (mM) pH (mM) (%) incorporated) As shown in Figure 3, within these ranges, Taq showed an improved fidel- A. Initial conditions ity when the pH was reduced to 8. Sequenase 2.55 8.0 5 90 5.4 • 10-5 Vent showed no change in fidelity Taq 0.2 8.8 2 70 2.0 • 10 -4 from pH 8 to 9, but fidelity decreased Vent 0.2 8.8 2 70 6.6 x 10 -5 at pH 7. The fidelity of Sequenase was B. Improved conditions invariant over the pH range of 7 to 9. Based on these observations, pH 8.0 Sequenase 3.5 8.0 2.5 90 4.4 x 10-5 Taq 0.5-1.5 8.0 5 36 7.2 x 10-5 was used for all three polymerases for Vent (and Rec-Vent) 0.5-1.5 8.5 7.5 70 4.5 • 10-5 the next step of the optimization procedure.

PCR Methods and Applications 65 Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

dNTP concentrations were varied at the pH and MgSO 4 concentrations that i, I,,,,,,~'- 20 optimized fidelity. As seen by compari- 0 j-~ Sequenase u i son with Figure 2, Taq was now found r" to amplify at dNTP concentrations 15 above 0.5 mM, albeit with low ef- /, ficiency (-36%). Remarkably, at pH 8 and 5 mM MgSO4, the error rate of Taq dropped toward that seen previously

" 0 for Vent and Sequenase, reaching a ,-- 5 0"-..0 ..-0 maximum fidelity at or above 0.5 mM 0 lb.. L.. dNTP. This effect was not found with

| 0 ,., ,., , ,,,,.,.,.t/ the first round of optimizations (Figs. 0.0 0.2 0.4 0.6 0.8 1.0 2 3 4 2, 3, and 4), and emphasized the prac- Each dNTP (mM) tical importance of reexamining the effect of the first two factors, namely pH FIGURE 2 Fidelity of three DNA polymerases as a function of equimolar dNTP concentra- and dNTP (Figs. 5 and 6). tions: (&)Taq (pH 8.8, 2 mM MgSO4) , (I-1)Vent (pH 8.8, 2 mM MgSO4) , and (O)Sequenase (pH The fidelity of both Vent and Rec- 8.0,5 mM MgC12). The efficiency was -90% for Sequenase, -70~ for Taq, and 70% for Vent, Vent was relatively invariant with for all dNTP concentrations shown. The arrows indicate the initial concentration of dNTP. dNTP concentration (Fig. 6), but once again a difference between Vent and The next factor studied was the by comparing Figure 5 with Figure 3, Rec-Vent preparations was found in concentration of magnesium ion. Two the fidelity of Taq as a function of pH that only Vent showed efficient ampli- separate batches of Taq (Perkin Elmer was not changed by the alterations in fication below 0.5 mM dNTP. Because Cetus) were used. One batch of Taq dNTP or MgSO 4 concentrations. The only Rec-Vent will be commercially showed acceptable amplification best fidelity of Taq was still obtained at available, except upon special request (-35%) only from 5 to 10 mM Mg. As pH 8 and efficiency dropped off below from the manufacturer, the Rec-Vent shown in Figure 4, with the mag- pH 8. However, the shift in dNTP and results should be noted in standard nesium concentrations that permitted MgSO 4 concentrations did eliminate PCR applications. acceptably efficient amplification, the the increased error rate of Vent fidelity of Taq was improved both by polymerase observed at pH 7 in Figure DISCUSSION increasing MgSO 4 to 10 mM or decreas- 3. Now a difference between native The error frequency of different DNA ing it to 0.5 mM. Vent polymerase and Vent and recombinant Vent polymer- polymerases has been measured by a its bacterially produced cloned version, ase was found: The efficiency of Rec- variety of assays and ranged from 10 -4 Rec-Vent, were found to have similar Vent dropped precipitously below pH to 10 -7 error/base pair incorporat- efficiency and fidelity. Their efficien- 7.5. ed. 14-7) In this study, a combination of cies were insensitive to MgSO 4 con- Figure 6 shows the fidelity of Taq, PCR and DGGE provides a sensitive centrations over the range studied, but Vent, and Rec-Vent polymerases as and reliable assay to measure the wide below 2 mM MgSO4, the fidelity of Vent and Rec-Vent DNA polymerase decreased drastically. A small but use- 9u 25 ful improvement in Sequenase fidelity occurred when MgC12 was decreased L,, 0 from 5 mM (Table 1A) to 2.5 mM. "L. 20 ty,a ~ 0 At this point conditions had been U defined which yielded fidelities about C 15 4.5 x 10 -s for both Vent and Sequenase .__ m and as low as about 1.2 x 10 -4 for Taq polymerase, each representing im- ~ 10 provement of fidelity over that obtained using the original conditions of Sequenase;equ~)ntse0 .... i ,- 5 Table 1A. Vent To test for covariance of effects on 0 fidelity among the variables studied, | 0 I ' I ' I ' I ' I ' the lowest error rate conditions from 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Figure 4 were then probed in reverse pH order with regard to the effects of pH FIGURE 3 Fidelity of Sequenase, Taq, and Vent DNA polymerases as a function of pH: (~k)Taq and then dNTP using the two thermo- (0.05 mM each dNTP, 2 mM MgSO4) , (I--I)Vent (0.5 mM each dNTP, 2 mM MgSO4),(O) Se- stable polymerases, Vent (Rec-Vent) quenase (3.5 rnM each dNTP, 5 mM MgCl2). The efficiency was -90% for Sequenase, -70% for and Taq polymerases. As may be seen Taq, and Vent, except for Vent at pH 9 where it was -45%. The arrows indicate the initial pH.

66 PCR Methods and Applications Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

"0 equimolar concentration of total dNTP 16- to Mg 2§ concentration and that, in I,. general, it is improved by lowering ~ 14 dNTP concentration, even in excess of u Mg2§ (7,9) Our results confirm these " 12 " Taq reports but also show that for both Taq and Sequenase, in conditions where \ the Mg 2§ concentration is high and the ~ a " .4~ Sequenase total dNTP concentration is less than ~ 6 that of magnesium, fidelity is optimal o ...... 9-"-'~"=~""" ...... "xRec'Vent 9 Vent at higher dNTP concentration. How- ~, 4 ever, the fidelity of Vent, though in- L. variant over a wide range of dNTP con- | 2 I ' I I ' I ' I 0 2 4 6 8 10 12 centration, slightly decreased at high dNTP concentration. This may be due Magnesium (mM) to diminished proofreading ability by FIGURE 4 Fidelity of Sequenase, Taq, Vent, and Rec-Vent as a function of Mg 2§ concentra- high dNTP concentrations that has tions:(A k)Taq (0.05 mM each dNTF),(I'-I 9 X )Rec-Vent (both 0.5 mM each dNTP), and been observed in many DNA polymer- (O)Sequenase (3.5 mM each dNTP). All reactions were at pH 8.0, using MgC12 for Sequenase ases with 3'-.5 ' exonuclease activity and MgSO 4 for Taq, Vent, and Rec-Vent. The efficiency was 90% for Sequenase, -70% for T, lq such as E. coli Pol I and T4 polymer- Lot 3813(~I, ),except at <1 mM Mg 2+ (36%), 40-63% for Taq Lot 3964( A),and -70% for Rec- ase. (6,17,23,24) High concentrations of Vent and Vent, except at 0.5 rnM Mg 2§ (60%). The arrows indicate the initial Mg 2§ con- dNTP are postulated to increase the centration. rate of polymerization of the next correct nucleotide following the mispair, and thus decrease the probability of range of fidelity of different DNA poly- have an active 3 ' -*5 ' exonuclease ac- excision by the exonuclease. (23'2s) merases over a variety of sequence con- tivity. However, its fidelity was texts in the 104-bp target studied. (4) matched by Sequenase and was 1.6- The effect of pH again showed no In its present form, error rates as low as fold better than Taq polymerase, both common trend on the fidelity of the 5 x 10 -6 errors/base pair incorporated of which are reported to be devoid of three DNA polymerases tested. There can be measured if care is taken to be- exonuclease activity. (8,22) was only a slight effect on the fidelity gin with a sufficiently large number of The fidelity of all three enzymes, of Sequenase. The fidelity of Vent denaturing gradient gel-purified Sequenase, Taq, and Vent, was shown decreased at low pH only in conditions templates to eliminate jackpot effects. to be sensitive to changes in pH, con- of low equimolar concentrations of Another advantage is that DGGE pro- centrations of dNTP, and Mg 2§ ions. dNTP to Mg 2§ whereas the fidelity of vides a fast and accurate way to The fidelities of Taq and Sequenase Taq progressively decreased as pH was measure polymerase-induced mutants were shown to have a different depen- raised. This supports the results of pre- in virtually any sequence of interest to dence on dNTP concentration than vious studies on Taq, (7,9) that at all Mg 2§ and dNTP concentrations, the fi- the investigators. All base-pair substitu- Vent. Previous studies of Taq had con- delity of can be improved by tions, small additions, and deletions cluded that its fidelity is good at low Taq are detected. The nature of the sequence changes can be determined by isolating and sequencing these DNA -o 25 O fragments. (4,13,21) However, if such in- 0o formation is not required, relative O 20 y Taq changes in the nature of polymerase- O induced errors can be inferred from the c pattern on the denaturing gradient gel ._ 15 alone. The fidelity of DNA synthesis depends on the ability of DNA polymerases to discriminate against an in- ~]Vent Rec-Ven correct nucleotide being ligated onto a 9-- 5

0 primer-template, to extend from the L.. L. mispair, and to correct the mispair by Q 0 ' I ' I " I 9 I ' I ' 3'--5' exonucleolytic reversal of 6.5 7.0 7.5 8.0 8.5 9.0 9.5 polymerization. This last ability is pos- pH sible only for DNA polymerases with active 3 ' -*5 ' proofreading exonucle- FIGURE 5 Fidelity of Taq, Vent, and Rec-Vent as a function of pH:( 9)Taq (0.05 mxl each ase. Among the three enzymes studied dNTP, 5 mM MgSO4),( [] )Vent and( x )Rec-Vent (both 0.5 mM each dNTP, 7.5 mM MgSO4). here, Vent is the only one known to The efficiency was -40% for Taq, -70% for Vent, and Rec-Vent (57% at pH 9).

PCR Methods and Applications 67 Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

9u 25 o from the U.S. Department of Energy (DE-FG02-86ER60448) and the Nation- 0 ~ 2O al Institute of Environmental Health 0 u Sciences (P30-ES02109, 2P01-ES03926, c 5P01-ES01640, and 5P42-ES04675). ._. 15 REFERENCES 1. Saiki, R.K., S. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Erlich, and N. Arnheim. 1985. Enzymatic amplifica- ~ 5 ]>r '-' Rec-Vent L. [• tion of B-globin genomic sequences 0 I,.,. and restriction site analysis for 0 0 ' I ' I I diagnosis of sickle cell anemia. Science 0.0 0.5 1.0 1.5 230: 1350-1354. each dNTP (mM) 2. Mullis, K.B. and F.A. Faloona. 1987. Specific synthesis of DNA in vitro via FIGURE 6 Fidelity of Taq, Vent, and Rec-Vent as a function of dNTP concentrations:( A )Taq a polymerase-catalysed chain reac- (5 mM MgSO4, pH 8.0),( [] )Vent, and( x )Rec-Vent (both 7.5 mM MgSO4, pH 8.51. The ef- tion. Methods Enzymol. 155: 335-350. ficiency was 34-38% for Taq, except at 0.5 and 1.5 mM dNTP (-59%), and -70% for Vent and Rec-Vent. 3. Kleppe, K., E. Ohtsuka, R. Kleppe, I. Molineux, and H.G. Khorana. 1971. Studies on polynucleotides. XCVI. lowering the pH. Mg 2§ is used in this ted and are being sequenced. Repair replications of short synthetic study as the activating divalent metal The fidelity of Sequenase, Taq, and DNA's as catalyzed by DNA polymer- ion for DNA polymerization. It is con- Vent can be usefully improved by ap- ases. J. Mol. Biol. 56: 341-361. sidered a physiological activator be- propriate changes in pH, concentra- 4. Keohavong, P. and W.G. Thilly. 1989. cause it is found in cells.(s) Depending tions of dNTP, and Mg 2§ ion relative to Fidelity of DNA polymerases in DNA on its concentration, Mg 2§ has been conditions previously employed in amplification. Proc. Natl. Acad. Sci. shown to increase or decrease polymer- PCR. It is probable that there are fac- 86: 9253-9257. ase fidelity. (7,9,18,]9) Fidelity has been tors other than dNTP, pH, and Mg 2§ 5. Loeb, L.A. and T.A. Kunkel. 1982. shown to decrease by increasing the that are also important for their fidel- Fidelity of DNA synthesis. Annu. Rev. concentration of free Mg2+. (7,9) Indeed, ity in which overall mutant fractions Biochem. 52: 429-457. the fidelities of Sequenase and Taq of less than 10% are required for 100- 6. Kunkel, T.A., L.A. Loeb, and M.F. were very slightly decreased by high bp sequences amplified a million-fold. Goodman. 1984. On the fidelity of concentrations of Mg 2§ The high fidel- The conditions represented in the DNA replication. J. Biol. Chem. 259: ity of Vent was mainly invariant and results of this study were limited to 1539-1545. decreased only at very low Mg 2§ con- those that allowed efficient amplifica- 7. Eckert, K.A. and T.A. Kunkel. 1990. centration. Other studies on DNA tion of a specific template, HPRT exon High fidelity DNA synthesis by the polymerases with 3'--,5' exonuclease 3. It is probable that fidelity and ef- Thermus aquaticus DNA polymerase. activity such as E. coli Pol I have ficiency are functions of the DNA se- Nucleic Acids Res. 18: 3739-3744. similarly noted that there was no effect quence being amplified. The variations 8. Tindall, K.R. and T.A. Kunkel. 1988. of Mg 2§ concentration on their fidel- in the fidelity and efficiency of dif- Fidelity of DNA synthesis by the ity, (18) except at inhibiting concentra- ferent lots of DNA polymerases is of Thermus aquaticus DNA polymerase. tion.(19) practical importance in experimental Biochemistry 2 7:6008-6013. As adjustments in the PCR condi- procedure and should be noted. Thus, 9. Eckert, K.A. and T.A. Kunkel. 1991. tions were made, the intensities of all it is important to optimize the PCR The fidelity of DNA polymerases used of the heteroduplexes relative to the conditions based on the DNA sequence in the PCR. In Polymerase chain WT homoduplex changed. However, of interest and the purpose of the reaction: A practical approach (eds. M.J. there was no observable change in the study. The major improvement in the McPherson, P. Quirke, and G.R. bands of the heteroduplexes in the fidelity of Taq was achieved at very low Taylor), pp. 227-246. IRL Press at denaturing gradient gel. This indicates amplification efficiency (-36%) where- Oxford University Press, Oxford (in that hotspots for polymerase-induced as Vent (-70%) and Sequenase (-90 %) press). errors were similar regardless of consistently showed efficient amplifi- 10. Scharf, S.J., G.T. Horn, and H.A. changes in the PCR conditions em- cation. Our results showed that Vent, a Erlich. 1986. Direct cloning and ployed. The specific polymerase- thermostable DNA polymerase, is as ac- sequence analysis of enzymatically induced mutants of Sequenase and Taq curate in DNA synthesis as Sequenase amplified genomic sequences. Science have been previously reported.(4) The and should be of use to researchers 233: 1076-1078. positions of the heteroduplexes in the generating PCR products. 11. P~i~ibo, S., D.M. Irwin, and A.C. denaturing gradient gel are very similar Wilson. 1990. DNA damage promotes between Taq and Vent, with a few ex- ACKNOWLEDGMENTS jumping between templates during ceptions. These bands have been isola- This work was supported by grants enzymatic amplification. ]. Biol.

68 PCR Methods and Applications Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Chem. 265: 4718-4721. Exonucleolytic proofreading en- 12. Gyllensten, U. and H.A. Erlich. 1988. hances the fidelity of DNA synthesis Generation of single stranded DNA by chick embryo DNA polymerase-u by the polymerase chain reaction and I. Biol. Chem. 263: 4450-4459. its application to direct sequencing of 25. Ninio, J. 1975. Kinetic amplification the HLA DQ alpha locus. Proc. Natl. of enzyme discrimination. Biochimie Acad. Sci. 85: 7652-7656. 57: 587-595. 13. Cariello, N.F., J.K. Scott, A.G. Kat, W.G. Thilly, and P. Keohavong. 1988. Received May 1, 1991; accepted in revised Resolution of a missense mutant in form July 2, 1991. human genomic DNA by denaturing gradient gel electrophoresis and direct sequencing using in vitro DNA amplification: HPRTMunich. Am. J. Hum. Genet. 42: 726-734. 14. Keohavong, P., A.,G. Kat, N.F. Cariello, and W.G. Thilly. 1988. Laboratory methods: DNA amplification in vitro using T4 DNA polymerase. DNA 7: 63-70. 15. VentTM DNA polymerase technical bulletin from New England Biolabs. 1990 and 1991. 16. Goodman, M.F. 1988. DNA replication fidelity: Kinetics and thermodynamics. Mutat. Res. 200: 11-20. 17. Kunkel, T.A., R.D. Sabatino, and R.A Bambara. 1987.Exonucleolytic proofreading by calf thymus DNA polymerase delta. Proc. Natl. Acad. Sci. 84: 486S-4869. 18. Sirover, M.A., D.K. Dube, and L.A. Loeb. 1979. On the fidelity of DNA replication: Metal activation of Escherichia-Coli DNA polymerase. I. Biol. Chem. 254:107-111. 19. Kunkel, T.A. and L.A. Loeb. 1979. On the fidelity of DNA replication: Effect of divalent metal ion activators and deoxyribonucleoside triphosphate pools on in vitro mutagenesis. J. Biol. Chem. 254: 5718-5725. 20. Weymouth, L.A. and L.A. Loeb. 1978. Mutagenesis during in vitro DNA synthesis. Proc. Natl. Acad. Sci. 75: 1924-1928. 21. Myers, R.M., N. Lumelsky, L.S. Lerman, and T. Maniatis. 1985. Detection of single base substitution in total genomic DNA. Nature 313: 495-498. 22. Huber, H.E., M. Russel, P. Model, and C.C. Richardson. 1986. Interaction of mutant thioredoxins of Escherichia coil with the gene 5 protein of phage T7.1. Biol. Chem. 261: 15006-15012. 23. Kunkel, T.A. 1988. Exonucleolytic proofreading. Cell 53: 837-840. 24. Kunkel, T.A. and A. Soni. 1988.

PCR Methods and Applications 69 Downloaded from genome.cshlp.org on October 2, 2021 - Published by Cold Spring Harbor Laboratory Press

Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and vent DNA polymerases.

L L Ling, P Keohavong, C Dias, et al.

Genome Res. 1991 1: 63-69 Access the most recent version at doi:10.1101/gr.1.1.63

References This article cites 23 articles, 12 of which can be accessed free at: http://genome.cshlp.org/content/1/1/63.full.html#ref-list-1

License

Email Alerting Receive free email alerts when new articles cite this article - sign up in the box at the Service top right corner of the article or click here.

To subscribe to Genome Research go to: https://genome.cshlp.org/subscriptions