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An Efficient and Optimized PCR Method with High Fidelity for Site-directed Mutagenesis

Qianwa Liang, Lisha Chen, and Armand J. Fulco

Department of Biological Chemistry and the Laboratory of Structural Biology and Molecular Medicine, School of Medicine, University of California, Los Angeles, California 90024-1737

We have developed an efficient provements in efficiency, flexibility, round of PCR amplification, the muta- method for site-directed mutagene- and fidelity. genic primer and the antiparallel univer- sis using two subsequential rounds of sal primer are used to produce a specific PCR. In this method, PCR conditions DNA fragment carrying the mutation. are optimized to favor high fidelity of This DNA fragment is purified and then Taq DNA polymerase in the presence PCR with Taq DNA polymerase has used as a primer together with the sec- of equimolar concentrations of been widely used for both the amplifica- ond universal primer for the second MgCI 2 and dNTP in the reaction mix- tion of specific DNA sequences and site- round of PCR amplification. However, a ture (pH $.5--6.2). This method directed mutagenesis. In the last 6 years, frequent drawback of this method, as makes use of a pair of universal prim- two major methods for site-directed mu- pointed out by Barettino et al., ~4~ is inef- ers and the multiple cloning site of tagenesis using two subsequential ficient priming by the megaprimer frag- pUC/M13 vectors. Only one muta- rounds of PCR amplification have been ment in the second round of PCR ampli- genic primer is required per target developed. The first method, designated fication, which results in very low yields site. In the second round of PCR, the overlap extension, was described by Ho of the full-length fragment of target 3' extension of the wild-type DNA et al. (1~ In this method a pair of overlap- DNA. Recently, several different ap- strand is blocked by the presence of a ping mutagenic primers and two flank- proaches for improving the megaprimer segment of nonhomologous se- ing primers are required. With two dif- method have been described (4's~ but, as quence at its 3' end, and as a conse- ferent combinations of one flanking the investigators, note, these approaches quence, the amplified, full-length primer and one mutagenic primer, two introduce their own limitations. More- DNA fragment is chiefly from the mu- DNA fragments having overlapping ends over, in reviewing the various proce- tant strand. Furthermore, because are generated by two separate PCR am- dures for PCR-mediated site-directed the mutated DNA fragment has plifications. These fragments are com- mutagenesis published to date, we found flanking restriction sites different bined in a subsequent "fusion" reaction that little attention had been paid to the from those of the wild-type DNA frag- in which the overlapping ends anneal to optimization of mutagenic PCR with re- ment, the wild-type DNA fragment is each other, allowing the 3' overlap of gard to the fidelity of Taq DNA poly- totally excluded in the step involving each strand to serve as a primer for the 3' merase. Should DNA synthesis by Taq selective cloning of the mutant DNA extension of the complementary strand. DNA polymerase, used for this purpose, fragment. This method was success- The resulting fusion product is amplified be carried out under low-fidelity reac- fully used to introduce four, nonad- further by PCR. In practice, however, we tion conditions, the occurrence of un- jacent mutations in the S' regulatory found that the annealing of overlapping desired mutations would become a ma- region of the cytochrome P450SM_~ ends often fails to occur, which may be jor concern. gene. All 20 analyzed clones from attributable to the complementary reas- On the basis of the megaprimer strat- these four cases of mutagenesis car- sociation of double-stranded DNA in the egy, we developed an efficient method ried the desired mutations, and no fusion reaction and the adoption of sec- for site-directed mutagenesis using PCR undesired mutations were observed. ondary structure in single-stranded DNA for the introduction of multiple desired We observed that the larger the num- produced in the subsequent PCR. The mutations in the 5' regulatory region of ber of mismatched nucleotide resi- second method termed megaprimer, was the cytochrome P450BM_3 gene. There are dues in the mutagenic primer, the developed by Landt et al., (2~ based on the three major advantages in the method higher the concentration of MgCI 2 scheme described by Kammann et al. ~3) that we present here. First, the extension was necessary for successful PCR am- This method requires just one muta- of the 3' end of wild-type DNA strand is plification. Our experimental results genic primer and two flanking universal blocked by the introduction of a seg- indicate that this method offers im- primers for pUCfM13 vectors. In the first ment of nonhomologous DNA sequence

4:269-2749 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/95 $5.00 PCR Methods and Applications 269 Downloaded from genome.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press

to its 3' end so that the full-length frag- into plasmid vector pTZ19R to yield a re- RESULTS ment produced by the second round of combinant plasmid designated pFL3-1. Optimization of Reaction Conditions PCR amplification is mainly from the 3' To introduce site-directed mutations in extension of the mutant strand. Second, the regulatory region, the first round of for Mutagenic PCR the mutated DNA fragment can be selec- PCR was set up in a total volume of 100 Minimizing the frequency of undesired tively cloned into a plasmid vector by I~l containing 1 Izl of pFL3-1 (-5 ng), 20 mutations in PCR-mediated site-directed using appropriate flanking restriction pmoles of each primer, 10 ixl of 10x re- mutagenesis can be achieved by maxi- sites, as the mutated DNA fragment has action buffer [500 mM KCI, 100 mM Tris- mizing the fidelity of the DNA poly- flanking restriction sites different from HCl (pH 9.0), 1.0% Triton X-100], and merase. The error rate of Taq DNA poly- those of the wild-type DNA fragment. Fi- 2.5 units of Taq DNA polymerase; opti- merase used in the PCR can be nally, the conditions of PCR for site-di- mized amount of equimolar concentra- influenced by many factors in the reac- rected mutagenesis have been optimized tions of MgCl z and dNTP (see Optimiza- tion; (1~ major factors include MgC12 with regard to fidelity of Taq DNA poly- tion of Reaction Conditions for level, dNTP concentration, and pH con- merase to minimize undesired muta- Mutagenic PCR, below) were added. The ditions. Eckert and Kunkel(11)evaluated tions. reaction mixtures were overlayed with these factors extensively in PCR and re- 50 Ixl of light mineral oil, and the reac- ported that high fidelity of Taq DNA MATERIALS AND METHODS tion was carried out in an automatic polymerase could be reached when thermal cycler for four cycles of 2 min at MgC12 and dNTP were present at equi- Materials 94~ 1 min at 48~ and 2 min at 72~ molar concentration or at a pH between Restriction endonucleases, T4 DNA li- and then for 20 cycles of 1 min at 92~ 5 and 6 (70~ at 1.5 mM MgC1 z. Because gase, and T4 DNA polymerase were pur- 1 min at 58~ and 2 min at 72~ The the PCR mixtures, when set up with the chased from either New England Biolabs 72~ incubation of the last cycle was ex- 10x reaction buffer supplied with Taq or GIBCO BRL. Taq DNA polymerase was tended for an extra 5 min before the re- DNA polymerase from Promega, gave a purchased from Promega. Oligonucle- action samples were cooled to room pH of 5.5-6.2 at 70~ our efforts to op- otide primers used in PCR and DNA se- temperature. The second round of PCR timize the mutagenic PCR conditions quencing were synthesized by Integrated amplification was set up as described with regard to the fidelity of Taq DNA DNA Technologies, Inc. Deoxynucle- previously, (7) except 2 mM MgClz and 0.5 polymerase were focused on the adjust- otide triphosphates (dNTPs) and mM each of the four dNTPs were used ment of the MgClz and dNTP concentra- [~-3SS]dATP were from Amersham Corp., here. The PCR was performed for 20 cy- tions. Mutagenic primers and pUC/M13 the Geneclean kit was purchased from cles, each cycle consisting of 92~ for 1 universal primers used for the introduc- Bio 101, Inc., and the Sequenase kit and min, 57~ for 1 min, and 72~ for 2.5 tion of mutations in three different loca- plasmid pTZ19R were from U.S. Bio- min. The last cycle was followed by 72~ tions of the 5' regulatory region of the chemical Corp. The 5' regulatory region for 5 min before cooling to room tem- P450BM_~ gene are shown in Table 1. The of the cytochrome P450BM_3 gene in Ba- perature. mutagenic primers carry one to nine cillus megaterium ATCC 14681 was mutant nucleotide residues. Because the cloned in our laboratory. (6) The DNA se- error rate of DNA synthesis by Taq DNA polymerase in the PCR has been shown quence of the P450BMo3 gene, including General DNA Methods the 5' regulatory region, is available un- to increase with increasing MgCl 2 con- der GenBank accession number J04832. DNA cloning, plasmid isolation, restric- centration (dNTP at 1 mM), O1) the min- Escherichia coli strain JM109 was used as tion enzyme digestion, and gel electro- imum concentration of MgClz required a host for plasmid transformation and phoresis were performed according to for efficient mutagenic PCR amplifica- preparation. All chemicals used in the current procedures. (8) Recovery of DNA tion is the optimum level of MgC1 z in experiments were reagent grade or bet- fragments from low-melt agarose gel the reaction mixture for the purpose of ter. with the Geneclean kit was carried out minimizing undesired mutations. PCR following the protocols supplied by the was set up with 1-4.5 mM MgC12 for each manufacturer. DNA sequencing analysis pair of primers, with the results shown PCR was performed by the dideoxy method (9) in Figure 1. In PCR with a pair of univer- The 5' regulatory region (1.63 kb) of the using pUC/M13 universal primers and sal primers, forward primer (pUC/M13-F) cytochrome P4508M_3 gene was cloned P450BM_3-specific synthetic primers. and reverse primer (pUC/M13-R), the tar-

TABLE 1 Two Universal and Three Mutagenic Primers Used for Site-directed Mutagenesis by PCR in the 5'-Regulatory Region of the Cytochrome P450BM_S Gene of Bacillus megaterium

Primer Designation Sequence Mismatches (bp) Product size (bp)

pUC/M13-F 5-CGCCAGGGTTTTCCCAGTCACGAC-3r r 0 pUC/M13-R 5'-TCACACGGAAACAGCTATGACC-3' 0 1780 JVmz 5-ATTGTTCCTGCTTCTACTTTGGC-3t i 1 1163 BB3mz 5'-AAATTCCACCAGTTTCTGATATGC-3' 2 1418 Oiiim2 5'-CTTTTCAAAAACAGTAACATCCTGAACTCCGCTACAATTAC-3' 9 1025

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with pUC/M13-F and mutagenic primer JVm2, BB3m2, and OiIim2, respectively. The corresponding equimolar dNTP concentrations in the reactions for these three mutagenic PCR runs were 0.5, 0.75, and 1.0 mM, respectively, of each of the four dNTPs (i.e., 2, 3, and 4 mM total).

Procedures for Site-directed Mutagenesis Using PCR The strategy of the PCR method for site- directed mutagenesis is outlined in Fig- ure 2. The P450BM_3 promoter region of the 1.6-kb DNA fragment was cloned into pTZ19R through the unique SalI and BamHI sites at the multiple cloning site of the vector. The resulting construct contained multiple unique restriction sites at both flanking ends of the insert. The first round of PCR was mutagenic, using flanking primer pUC/M13-F and the mutagenic primer m2. The reaction was performed under the optimized con- ditions to favor the high fidelity of Taq DNA polymerase activity as described above. The specifically amplified DNA fragment was purified by low-melting agarose gel electrophoresis and the Geneclean kit. In the interim, the flank- ing restriction site "A" of the insert in the plasmid was replaced by restriction site "C" (HindIII in our examples) by DNA manipulation. In our experiments, the KpnI site served as the A site. A 12-bp HindIII linker with the 5' end phospho- rylated was used as the C site to replace the KpnI site. Following KpnI restriction FIGURE 1 The minimal requirement of MgCl 2 for each mutagenic PCR is determined by mon- enzyme digestion, the linearized plas- itoring the PCR-amplified target DNA by electrophoresis on an agarose gel. Each primer combi- mid was treated with T4 DNA polymerase nation and its corresponding amplified DNA fragment, as analyzed by agarose gel electrophore- in the presence of dNTP to generate sis, are placed side by side. PCR samples on all four gels have the same lane alignments. For each blunt ends and then purified using PCR reaction (100 I~l total), 10 I~l of sample was loaded onto the gel. (Lane 1) DNA molecular Geneclean. Next, the linear plasmid was weight standards; (lanes 2-9) results of eight different PCR samples with a range of 1-4.5 mM MgC12 in the reaction mixtures (see the plot, top). The sizes of the standards (left) and those of recircularized with the HindlII linker us- specific amplified DNA fragments (right) are shown on the sides of the electrophoretographs. ing DNA ligase. The resulting plasmid then was linearized by digestion with re- striction enzyme C and purified by using low-melting agarose gel electrophoresis get DNA fragment (1.78 kb) was signifi- matched nucleotides) specific PCR am- (to remove undigested circular plasmid) cantly amplified when the MgC12 con- plification was not observed until the and Geneclean. The linearized plasmid centration was ~1.5 mM. However, with MgC12 in the reaction mixture reached a and the PCR-amplified DNA fragment mutagenic primers, the PCR amplifica- concentration of 3 mM. For pUC/M13-F containing the desired mutations (-5 ng tion needed higher concentrations of and mutagenic primer Oiixm2 (nine mis- for each preparation) were then com- MgC12. For example, in the PCR with matched nucleotides), the minimal bined and used as templates for the sec- pUC/M13-F and mutagenic primer JVm2 MgC12 concentration for significant PCR ond round of PCR amplification. As (which carried one mismatched nucle- amplification was 4 mM. Thus, 2, 3, and 4 shown in Figure 2, after the two DNA otide), the minimal concentration of mM MgC12 were used as the optimal fragments were added together in the re- MgC12 required for efficiency was 2 mM. MgC12 concentrations for the mutagenic action mixture, the single-stranded DNA For pUC/M13-F and mutagenic primer PCR (the first round of PCR in the pro- from the fragment of the first round of BB3m2 (which contained two mis- cedures as presented in the next section) PCR (together with the identical single-

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F A B T. m2 replace A site with R ~ C linker B PCR with F/m2/~~~]

cut with enzyme C I , ~

TI.

R PCR with F/R A B

cut with restriction enzyme A and B, cloning into plasmid vector. FIGURE 2 Diagrammatic scheme of the mutagenesis procedure. Only the insert (unfilled segment in diagram) and its flanking regions (shaded boxes) are shown here. F and R represent the pUCfM13 forward and reverse primers, respectively. The mutagenic primer is designated m2. A, B, and C represent three different flanking restriction sites. The 3' extension is indicated by a broken line. (/) The scheme for the first round of PCR (mutagenic); (I/) the scheme for the second round of PCR. stranded fragment from the first cycle of sert carrying the desired mutation was the JV site. In the first case, the muta- the second round of PCR as primed by then selected by digestion with restric- genic PCR was carried out with muta- pUC/M13-F) anneals to its complemen- tion enzymes A and B and recovered by genic primer JVml and pUCfM13-R (Fig. tary strand of the linearized plasmid low-melting agarose gel electrophoresis 3, lane 1). The flanking restriction site B DNA or to the single-stranded DNA gen- and Geneclean treatment. The resulting was replaced by C in the plasmid. After erated by the pUC/M13-R primer in the mutated DNA fragment was cloned into the combination of the mutated DNA PCR to form a heteroduplex. In this het- vector pTZ19R by using the same A and B fragment and the plasmid linearized by eroduplex, the 3' end of the mutant restriction sites. Because any wild-type the digestion with restriction enzyme C, strand DNA could be extended to the DNA fragments that might, by chance, the full-length target DNA fragment con- pUC/M13-R primer annealing site to be amplified in the PCR would carry taining the desired mutation was ampli- generate a full-length mutated single- flanking restriction sites B and C, they fied successfully in the second round of stranded insert. This single-stranded were excluded in the cloning step be- PCR (Fig. 3, lane 3). In the second case, DNA then became a homoduplex DNA cause the A and B restriction sites were the first round of PCR was carried out in the second cycle of the PCR primed by used for this process. Finally, the desired with mutagenic primer JVm2 and pUC! pUC/M13-R primer. In contrast, the 3' mutation in the insert may now be con- M13-F (Fig. 3, lane 2). The flanking re- end of the wild-type strand DNA of the firmed by DNA sequencing analysis. striction site A was replaced by C in the heteroduplex could not be extended be- plasmid, and the second round of PCR cause the 3' end (4 nucleotide residues also significantly amplified the target in our examples) was not homologous to DNA fragment (Fig. 3, lane 4). Our over- Mutagenesis of the P4$OsM_ 3 its template and the Taq DNA poly- all evaluation of these two alternative Promoter Region Using PCR merase, which lacks a 3'--> 5' exonu- ways for introducing mutations at the JV clease activity, was not capable of proof- Using PCR, we successfully introduced site, and the successful use of these pro- reading. ~ The homoduplex DNA desired mutations into four different lo- cedures for the introduction of four non- containing the desired mutation was cations on the 5' regulatory region of the adjacent mutations into the 5' regula- amplified by the two flanking universal P450BM_3 gene. Figure 3 shows the results tory region of the P450BM_3 gene, in- primers in the remaining cycles of the of PCR with two different primer combi- dicates that our PCR method is very flex- second round of PCR. The full-length in- nations for site-directed mutagenesis at ible in practice.

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here has been used successfully in our using our method, no undesired muta- laboratory for site-directed mutagenesis tions were observed among the analyzed on the 5' regulatory region of the cy- clones. In practice, high fidelity is very tochrome P450BM_3 gene in B. megate- important as researchers, after perform- rium. Our PCR method offers improve- ing site-directed mutagenesis on a cod- ments over other PCR mutagenesis ing sequence or a regulatory region us- techniques in efficiency, flexibility, and ing PCR, usually check only 200-300 bp fidelity. of sequence around the mutation site In this method wild-type parental rather than the whole length of inserted DNA is prevented from extension at the DNA. If there is an undesired mutation 3' end (Fig. 2) and is thus essentially ex- associated with the desired mutation on cluded from amplification during the the same DNA molecule, the effects of second round of PCR. Moreover, the mu- the desired mutation could be influ- tant DNA fragment generated by the sec- enced or even completely altered by the ond round of PCR amplification has undesired mutation. Some investiga- flanking restriction sites different from tors (14) have suggested using Vent DNA those of the wild-type parental DNA frag- polymerase instead of Taq DNA poly- ment; this allows for the selective clon- merase for site-directed mutagenesis us- ing of the mutant DNA fragment and, ing PCR, as the fidelity of Vent DNA poly- hence, exclusion of the wild-type DNA merase is 5- to 10-fold higher than that FIGURE 3 The PCR products of two alterna- fragment in the subsequent cloning observed for Taq DNA polymerase. (is) tive primer combinations for site-directed step. When we compared this method However, the 3' -~ 5' proofreading exo- mutagenesis on the JV site of the 5' regulatory with the megaprimer and overlap exten- nuclease activity of Vent DNA poly- region of P450~M_3 are shown after agarose gel sion methods for the introduction of merase may remove the mismatched nu- electrophoresis. Ten microliters of PCR sam- site-directed mutations at four different cleotide residues of the mutagenic ple was loaded in each lane. The amplified locations on the 5' regulatory region of primer and lead to a dramatically de- DNA products were visualized on the agarose the P450BM_3 gene, our procedure proved creased mutation efficiency. For exam- gels by ethidium bromide staining. (Lane 1) superior. For example, for mutagenesis Rxml and (lane 3) (Rxml)xF (lane 3) repre- ple, if used in our PCR method, the sent the first round and the second round of at the Oui site using the megaprimer 3'--> 5' proofreading activity of Vent PCR with a primer combination of pUC/M13- method, a very low yield in the second DNA polymerase would remove the 3' R, and JVml, respectively; (lane 2) Fxm2 and round of PCR amplification was ob- nonhomologous sequence of the wild- (lane 4) (Fxm2)xR represent the first round tained and only one of eight clones an- type DNA strand in the heteroduplex and the second round of PCR with a primer alyzed carried the desired mutation. and lead to amplification of parental combination of pUC/M13-F and JVmz respec- With the overlap extension method, DNA. An alternative approach would tively. The sizes of the standards (right) and only the desired mutations at the BB3 employ Vent (Exo-) DNA polymerase those of specific-amplified DNA fragments site were amplified in the second round (New England Biolabs), which has been (left) are shown at each side. of PCR; the other three cases of mu- genetically engineered to eliminate the tagenesis were unsuccessful. 3'---> 5' proofreading exonuclease activ- The basic strategy employed in our ity. (16) However, because the fidelity of method involves the use of a pair of uni- Vent (Exo-) DNA polymerase is only For each of these four cases of mu- versal primers, pUC/M13 forward (F) and twofold higher than that of Taq DNA tagenesis, five clones were picked ran- reverse (R) primers, which flank the in- polymerase, (is) we would still recom- domly for DNA sequence analysis. The serts in the pUC/M13 vectors. Only one mend optimization of the reaction con- results showed that all of the analyzed mutagenic primer is required per target ditions to maximize the fidelity of Vent clones (20 total) had the desired muta- site. As a consequence, this method, as (Exo-) DNA polymerase in PCR for site- tions. For each of the analyzed clones, demonstrated by our experiments pre- directed mutagenesis. With respect to -200 bp around the mutation site was sented here, is suitable for introducing optimization, the reason for the require- checked and no undesired mutation was multiple, nonadjacent mutations subse- ment of higher concentrations of MgC12 observed. In another experiment, the quentially on the same DNA molecule for PCR amplification with primers con- full length (1.63 kb) of the 5' regulatory with high efficiency. Furthermore, taining multiple mismatched nucleotide region of P4508M_3 gene containing mul- primer combinations can be changed at residues (Fig. 1) is not clear. It seems tiple mutations on three different loca- a mutation target site (Fig. 3), a flexibil- likely, however, that the mutagenic tions was checked by DNA sequencing ity in methodology that is especially im- primer/template hybrids need the pres- analysis. Again, no undesired mutation portant in cases where a specific primer ence of higher of Mg § + concentrations was observed. These results indicate that pair fails to work in PCR for undeter- to maintain their stability at high an- our PCR method introduces mutations mined reasons, as has been reported. (~3) nealing temperatures. very efficiently and with high fidelity. Our method also has the virtue of It should also be noted that Taq DNA high fidelity, as indicated by our mu- polymerase has been reported to cata- tagenesis results. In the introduction of lyze the untemplated addition of an A DISCUSSION four, nonadjacent mutations into the 5' (deoxyadenosine) residue at the 3' end The PCR method that we have described regulatory region of the P450BM_3 gene of the amplified DNA fragment. (w,~8)

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Mutagenic primers should therefore be 8. Sambrook, J., E.F. Fritsch, and T. Maniatis, designed as described (10) so that the first eds. 1989. Molecular cloning: A laboratory 5' nucleotide of a mutagenic primer fol- manual. Cold Spring Harbor Laboratory lows a T residue in the same strand of Press, Cold Spring Harbor, New York. 9. Sanger, F., S. Nicklen, and A.R. Coulson. template sequence. The amplified DNA 1977. DNA sequencing with chain-termi- fragment from the first round of PCR nating inhibitors. Proc. Natl. Acad. Sci. will thus have the correct sequence. 74: 5463-5467. When a T residue is not available before 10. Ling, L.L., P. Keohavong, Dias, C., and the first 5' nucleotide of a mutagenic W.G. Thilly. 1991. Optimization of the primer, the DNA fragment amplified by polymerase chain reaction with regard the first round of PCR must be treated to fidelity: Modified T7, Taq, and Vent with T4 DNA polymerase to remove the DNA polymerase. PCR Methods Applic. additional 3' residue before carrying out 1: 63-69. the second round of PCR amplification. 11. Eckert, K.A. and T.A. Kunkel. 1990. High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids ACKNOWLEDGMENTS Res. 18: 739-3744. 12. Tindall, K.R. and T.A. Kunkel. 1988. Fidel- We thank Keynes Tong from this labora- ity of DNA synthesis by the Thermus tory for his excellent technical assistance aquaticus DNA polymerase. Biochemistry in several of the experiments reported 27: 6008-6013. here. The research reported in this paper 13. Saiki, R.K. 1989. The design and optimi- was supported by National Institutes of zation of the PCR. In PCR technology (ed. Health Research grant GM23913 and by H.A. Erlich), pp. 7-16. Stockton Press, the Director of the Office of Energy Re- New York. 14. Watkins, B.A., A.E. Davis, F. Cocchi, and search, Office of Health and Environ- M.S. Reitz Jr. 1993. A rapid method for mental Research, contract DE-FCO3- site-specific mutagenesis using larger ER06015. plasmids as templates. BioTechniques 15: 700-704. 15. Mattila, P., J. Korpela, T. Tenkanen, and K. REFERENCES Pitkanen. Fidelity of DNA synthesis by the 1. Ho, S.N., H.D. Hunt, R.M. Horton, J.K. Thermococcus litoralis DNA polymerase-an Pullen, and L.R. Pease. 1989. Site-directed extremely heat stable enzyme with proof- mutagenesis by overlap extension using reading activity. Nucleic Acids Res. 19: the polymerase chain reaction. Gene 4967-4973. 77: 51-59. 16. Kong, H., R.B. Kucera, and W.E. Jack. 2. Landt, O., H.P. Grunert, and U. Hahn. 1993. Characterization of a DNA poly- 1990. A general method for rapid site-di- merase from the hyperthermophile ar- rected mutagenesis using the polymerase chaea Thermococcus litoralis. Vent DNA chain reaction. Gene 96: 125-128. polymerase, steady state kinetics, thermal 3. Kammann, M., J. Laufs, J. Schell, and B. stability, processivity, strand displace- Gronenborn. 1989. Rapid insertional mu- ment, and exonuclease activities. J. Biol. tagenesis of DNA by polymerase chain re- Chem. 268: 1965-1975. action (PCR). Nucleic Acids Res. 17: 5404. 17. Clark, J.M. 1988. Novel non-templated 4. Barettino, D., M. Feigenbutz, R. Valcarcel, nucleotide addition reactions catalyzed and H.G. Stunnenberg. 1994. Improved by procaryotic and eucaryotic DNA poly- method for PCR-mediated site-directed merase. Nucleic Acids Res. 16: 9677-9686. mutagenesis. Nucleic Acids Res. 22: 541- 18. Mole, S.E., R.D. Iggo, and D.P. Lane. 1989. 542. Using the polymerase chain reaction to 5. Marini, F. III, A. Naeem, and J.N. Lapeyre. modify expression plasmids for epitope 1993. An efficient 1-tube PCR method for mapping. Nucleic Acids Res. 17: 3319. internal site-directed mutagenesis of large 19. Kuipers, O.P., H.J. Boot, and W.M. de Vos. amplified molecules. Nucleic Acids Res. 1991. Improved site-directed mutagenesis 21: 2277-2278. method using PCR. Nucleic Acids Res. 6. Ruettinger, R.T., L.-P. Wen, and A.J. Fulco. 19: 4558. 1989. Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P450BM.3, a single peptide cytochrome Received October 11, 1994; accepted in P450:NADPH-P450 reductase from Bacil- revised form January 6, 1995. lus megaterium. ]. Biol. Chem. 264: 10987- 10995. 7. Liang, Q. and T. Richardson. 1992. A sim- ple and rapid method for screening trans- formant yeast colonies using PCR. Bio- Techniques 13: 730-735.

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An efficient and optimized PCR method with high fidelity for site-directed mutagenesis.

Q Liang, L Chen and A J Fulco

Genome Res. 1995 4: 269-274

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