Gette, 108 (1991) 1-6 8 1991 Elsevier Science Publishers B.V. All rights reserved. 0378-l 119/91/SO3.50 GENE 06172 High-fidelity amplification using a thermostable DNA polymerase isolated from P’~OCOCCUSfuriosus (Polymerase chain reaction; mutation frequency; lack; proofreading; 3’40-5 exonuclease; recombinant DNA; archaebacteria) Kelly S. Lundberg”, Dan D. Shoemaker*, Michael W.W. Adamsb, Jay M. Short”, Joseph A. Serge* and Eric J. Mathur” “ Division of Research and Development, Stratagene, Inc., La Jolla, CA 92037 (U.S.A.), and h Centerfor Metalloen~~vme Studies, University of Georgia, Athens, GA 30602 (U.S.A.) Tel. (404/542-2060 Received by M. Salas: 16 May 1991 Revised/Accepted: 11 August/l3 August 1991 Received at publishers: 17 September 1991 -.-- SUMMARY A thermostable DNA polymerase which possesses an associated 3’-to-5’ exonuclease (proofreading) activity has been isolated from the hyperthermophilic archaebacterium, Pyrococcus furiosus (Pfu). To test its fidelity, we have utilized a genetic assay that directly measures DNA polymerase fidelity in vitro during the polymerase chain reaction (PCR). Our results indicate that PCR performed with the DNA polymerase purified from P. furiosus yields amplification products containing less than 10s.~ of the number of mutations obtained from similar amplifications performed with Tuq DNA polymerase. The PCR fidelity assay is based on the ampli~cation and cloning of lad, lac0 and IacZor gene sequences (IcrcIOZa) using either ffil or 7ii~irqDNA poIymerase. Certain mutations within the lucf gene inactivate the Lac repressor protein and permit the expression of /?Gal. When plated on a chromogenic substrate, these Lacl - mutants exhibit a blue-plaque phenotype. These studies demonstrate that the error rate per nucleotide induced in the I82 known detectable sites of the lucl gene was 1.6 x lo-’ for Pfu DNA polymerase, a greater than tenfold improvement over the 2.0 x lo- 5 error rate for Tuq DNA polymerase, after approx. 105-fold amplification. INTRODUCTION porated nt (Tindall and Kunkel, 1988). The estimated error rate (mutations per nt per cycle) of Tuq polymerase varies The polymerase chain reaction (PCR) has become an from 2 x lo-” during PCR (Saiki et al., 1988; Keohavaong important tool in molecular biology. The automated in vitro and ?-hilly, 1989) to 2 x lo- 5 for nt substitution errors amplification process utilizes a thermostable DNA poly- produced during a single round of DNA synthesis of the merase; at present, the enzyme of choice is Tag DNA fucZa gene (Eckert and Kunkel, 1990). Such polymerase- polymerase (Mullis et al., 1986; Saiki et al., 1988). Purified induced mutations have hindered applications requiring Tbq DNA polymerase is devoid of 3’-to-5’ exonuclease high fidelity (Reiss et al., 1990; Ennis et al., 1990). (proofreading) activity and thus cannot excise misincor- P.,furiosus (‘rushing fireball’; DSM3638) was first iso- Correxpundenceio: Dr. E.J. Mathur, Stratagene, Inc., 11099 N. Torrey IPTG, isopropyl-/?-D-thiogalactopyranoside; kb, kilobase or 1000 bp; Pines Rd., La Jolla, CA 92037 (U.S.A.) l&02x, gene sequence of iacl, lac0 and 1acZx from E. coii; LB, Tel. (619)535-5400, ext. 5481; Fax(619)535-0071. Luria-Bertani (medium); mf, mutant frequency; nt, nucleotide(s); oligo, oligodeoxyribonucleotide; Pfu, Pyrococcus furiosus; PCR, polymerase Abbreviations: @Gal, &galactosidase (encoded by lac2); bp, base chain reaction; PNK, T4 polynucleotide kinase; Tug, Thermus u4~uijc~s; pair(s); BSA, bovine serum albumin; A, deletion; dNTP, deoxy- u, unit(s); wt, wild type; XGal, 5-bromo-4~chloro-3-indolyi-~-D-~alacto- ribonucleoside triphosphate; ER, error rate; EtdBr, ethidium bromide; pyranoside. 2 lated from geothermally heated marine sediments in Vul- (b) Proofreading ability of Pfu DNA polymerase cane, Italy (Fiala and Setter, 1986). This hyperthermophilic To analyze the 3’-to-5’ exonuclease activity at the archaebacterium grows optimally at 100°C by an unusual molecular level, we designed an assay to elucidate how a fermentative-type metabolism in which H, and CO, are the DNA polymerase responds to a mismatched 3’ terminus. only detectable end products (Adams, 1990). In this study, Three events can occur when a DNA polymerase interacts we demonstrate that a DNA polymerase isolated from with a mismatched 3’ primer terminus: no extension of the P. furiosus contains 3’-to-5’ exonuclease (proofreading) mismatched primer, extension with the incorporation of the activity and works effectively in PCR. Moreover. during mismatched nt, or extension following excision of the 3’ PCR, Pjii DNA polymerase exhibits eleven- to twelvefold mismatched nt. To address this question, a 35nt synthetic greater replication fidelity than 7’uq DNA polymerase. DNA template was constructed along with four 15-mer oligo primers complementary to the template but containing 0, 1, 2, and 3 mismatched nt at the 3’ termini (Fig. 1). The RESULTS AND DISCUSSION template was designed with an internal EcoRI site which coincides with the mismatched nt at the 3’ end of the (a) Py~ococcu~ DNA polymerase possesses 3’40-5’ exo- 15-mer primers. The 5’ end-labeled primers were annealed nuclease activity to the 35-mer template prior to incubation with either @u A thermostable DNA polymerase isolated from or Tuq DNA polymerase. Following extension, half of the P. ,furiosus was purified to greater than 99”/, homogeneity as reaction mixture was digested with EcnRI. f’fu DNA poly- visualized by polyacrylamide-gel electrophoresis. The merase extended all the primers tested, as demonstrated by purification and characterization of k’fuDNA polymerase the presence of 35-bp product before restriction digestion will be described elsewhere (E.J.M., in preparation). Taq (Fig. 1). In addition, Pfu DNA polymerase excised the mis- DNA polymerase was purchased from Perkin-Elmer/ matched nt of the primers and restored the EL.oRI site. as Cetus. Table I lists the polymerase and 3’-to-5’ exo- demonstrated by the digestion of 35-bp products. The prcs- nuclease-specific activities of @i and Tuq DNA poly- ence of degradation products (13- and 14-mers) seen with merases utilized in this study. some of the mismatched primers can be attributed to the active 3’-to-5’ exonuclease activity associated with Pfir TABLE I DNA polymerase. These reaction intermediates would not Specific activities of purified Pfu and Tuq DNA polymerases be detected if the extension reactions were to go to com- pletion. Taq DNA polymerase was capable of extending the Source of Protein Polymerase 3’ to 5’ wt control 15-mer primer to form a 35-bp product suscepti- DNA concentration.’ activityb cxonuclease ble to digestion with EcoRI, but the absence of labeled polymerase (mgiml) (uimg) activity’ (u/mg) 35-bp product in the reactions with the mismatched primers suggested that Tuq DNA polymerase was unable to extend P. furiosus 0.13 ( i 0.004) 31713 (2 1015) 9200 ( + 294) off any of these 3’ terminal mismatched primers (we chose T. aquaticus 0.30 ( f 0.015) 16667 (+ 850) negligible a G : G mismatch which is not efficient substrate for Tuq DNA polymerase). To fully characterize the proofreading I’ Protein determinations were performed by the method described by Bradford (1974). activity of a DNA polymerase, the composition and posi- b Polymerase activity assays were performed essentially as described by tion of all possible mismatches must be considered. The Grippo and Richardson (1971) except that the reaction mixture was assay described above was simply an initial screen for the incubated at 72°C instead of 37°C and that incorporated labeled nt were existence of proofreading activity in Pfu DNA polymerase. counted on DEAE filter disks (DE-8 I, Whatman), not by trichloroacetic acid precipitation, One unit of DNA polymerase activity catalyzed the incorporation of IO nmol total nt into a DEAE-bound form in 30 min at (c) PCR amplification with PfuDNA polymerase 72°C. Pfu DNA polymerase was also evaluated for use in PCR. ’ The exonuclease assay was performed essentially as described by Amplification reactions were performed with several differ- Chase and Richardson (1974) except that the reaction was incubated at ent primer : template combinations using either e/n or Tuq 72°C instead of 37’C and the substrate was 3’ labeled phage I DNA. Briefly, the substrate was prepared by digesting IOOpg phage i DNA DNA polymerase. In all cases tested, PCR performed with with 500 u Tuql restriction endonuclease in 100 mM K. acetate/25 mM l’fu DNA polymerase produced reaction products com- Tris acetate pH 7.6/10 mM Mg acetate/O.5 mM fi-mercaptoethanolj parable to those obtained with Tuq DNA polymerase. Four and 10 ki”g BSA per ml for 2 h at 65°C. This leaves dG and dC 5’ examples of amplification reaction products are shown in overhangs at 121 sites per phage I DNA molecule. These sites were Fig. 2. In most cases tested the specificity of amplification tilled-in with 50 u modified T7 DNA polymerase and excess [‘H]dGTP and [‘H]dCTP in 200 mM Tris HCI pH 7.5/100 mM MgClJ250 mM was increased with Pfu DNA polymerase. We do not yet NaCl at 37’C for 30 min. One unit of exonuclease activity catalyzed the know the reason for this, although i’/L DNA polymerase acid solubilization of 10 nmol of total nt in 30 min at 72-C. exhibits only half the activity of Trlq polymerase at room 3 (a) Flowchart (b) Proofreading Assay Y - TCCGGTCCC- -3 WI = 0 mismatch 5’ - TCCGGTCCC- -3’ ml = 1 mlsmalch Send label 5 - TCCGGTCCCGAATCG -3’ m2= 2 mismatch \ s - TCCGGTCCCGAAGCG -3’ m3= 3 mismatch L 15mer,,4 3’ - AGGCCAGGG-AGCAGCAGGCACGCGAACCG - 5’ 35mer template 2:’ EcoRl SW EcoRl de 35mer 5 -5’ end label 15mers with PNK’ -anneal rmsmat~hed 15mer to 35mer -anneal 15mer lo 35mer template -Incubate wfth DNA polymerase (1 1 molar rat10) m2 m3 15mer 15mer case3 extension.