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METHODS, AND APVU(ATIOH$ _A Highly Efficient Procedure for Site specific Mutagenes s of Full-length Plasmids Using Vent DNA Polymerase
Sridhar Byrappa, Denise K. Gavin, and Kailash C. Gupta 1
Rush-Presbyterian-St. Luke's Medical Center, Department of Immunology/Microbiology, Chicago, Illinois 60612
Careful titration of Vent polymerase stranded DNA is a cumbersome process. polymerase. Once standardized, the pro- activity allows efficient amplifica- Alternatively, site-specific mutants can cedure works consistently well. An addi- tion of full-length plasmids (12 kb). also be generated by amplifying mutant tional advantage to using Vent poly- The high processivity and fidelity of DNA employing two sequential rounds merase is that it generates blunt-ended this enzyme made oligonucleotide- of PCR from a double-stranded DNA products, ready for blunt-end ligation directed site-specific mutagenesis of template using 2 to 4 primers. (3-s) How- and cloning. In contrast, Taq poly- plasmids a straight-forward process. ever, in both cases, several experimental merase generates products with 3' over- Using only two primers, a mutagenic steps are required and the frequency of hangs requiring especially created vec- and a complementary, single-base obtaining mutants can vary from as low tors for cloning. mutants of recombinant plasmids as 0.1 to as high as 90%. Although we were obtained consistently with have efficiently created site-specific mu- MATERIALS AND METHODS >90% efficiency from a single round tants using single-strand DNA tem- of PCR. This procedure also made plates,
i Corresponding author. E-MAIL kgupta@rpslmc-edu; FAX (312) 226-6020.
404 ~ GENOME RESEARCH 5:404-407 ©1995 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/95 $5.00 Downloaded from genome.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press
PLASMID MUTAGENESIS
Table 1 Characteristics of PCR Primers Used for Site-specific Point Mutagenesis pcPC at 15, 20, 25, and 30 cycles using the reaction conditions described in Ma- GC terials and Methods. Analysis of reaction Primer Sequence Length Tm (%) Mutants a Frequency products (5 gl) revealed that whereas 15 cycles gave a distinct band of the appro- PI20 GGA CCG GGT OCC AGG CAC 18 79 77 4/4 100 priate size DNA, at 20, 25, and 30 cycles PI21 GCA CCG CTG AAT CGT TAC AAC 21 74 52 the synthesized DNA was progressively
P123 AAG GCA TCT TGA TCC ATG GT 20 70 45 6/6 100 degraded. Among the degradation prod- P122 CAT TCT AAA AGA AGA TTC TG 20 60 35 ucts were distinct bands of lower molec- ular weight DNA. We have no idea P124 CCG GGT GCC AGG CAC GGT 18 82 77 6/8 75 P125 GAC CCA CCG CTG AAT CGT TAC 21 75 57 about the basis of their origin. At the end of 30 cycles, a smear of heteroge- neous ~size DNA was visible (Fig. 1). Vent Bold and underlined nucleotides represent mutagenized sites. polyI~erase was used previously for site- aThe number of mutants obtained from the number of independent clones analyzed. specific deletion mutagenesis. (12) How- ever, when we used the recommended reaction conditions, the products were completely degraded. Clearly, inappro- plasmid DNA. At each PCR cycle, dena- added and the mixture was heated to priate optimization of amplification turation of DNA was carried out at 92°C 90°C for S min. Primers were used di- conditions results in degraded or inap- for 30 sec. The annealing temperatures rectly from this mixture for PCR reac- propriate sized products (Fig. 1). Analy- were generally 5 ° below the lower melt- tions. Primer sequences (Table 1) were sis of chain extension time revealed that ing temperature of the primer pair. determined by the requirements to gen- Vent polymerase under our reaction con- However, we observed that an annealing erate particular mutants rather than on ditions synthesized -1000 bp/min con- the guidelines used by many software temperature of 50°C worked well with sistent with a previous report. (7) Thus, all the primers we used (Table 1). Exten- programs to synthesize optimum prim- the vectors presented in this article re- sion time at 72°C was based on 1000 nu- ers. It is interesting to note that we had quired about 6 min of extension reac- cleotides/min as suggested by the Vent no problem using any primers irrespec- tion for each PCR cycle. We have also polymerase supplier and a previous re- tive of their sequences, melting temper- used Vent polymerase to extend a 12-kb port. {7) At the end of each PCR reaction, atures, and/or matching parameters. plasmid DNA without any problem an additional extension reaction of 10 However, wherever possible, we kept the (data not shown). Therefore, this proce- rain was carried out to complete synthe- melting temperature of the primer pairs dure permits direct amplification of an sis of any lagging chains. within 10°C. The melting temperatures entire plasmid of interest and its site- To obtain a sufficient amount of were determined using the Primer soft- specific mutagenesis. Recent success in ware (Scientific & Educational Software) DNA, three to five simultaneous reac- with the algorithm developed by Freier tions were carried out for each mutagen- et al. ~11) esis. The reactions were processed imme- diately at the end of the PCR cycles to minimize exonuclease activity. The M 15 20 25 30 PCR Reactions products were resolved in 0.7%-0.8% agarose gels in Tris-acetate-EDTA (TAE) As Vent polymerase has an efficient buffer. Appropriate bands were excised proof-reading 3' ~ S' exonuclease activ- from the gel and purified using Gene- ity, it degrades the synthesized DNA rap- clean (Bio 101). Purified DNA (100-200 6.7 kb- idly presumably after the DNA synthesis ng) was self-ligated and used to trans- precursors have been depleted. To over- form Escherichia coli HB101. For each come this problem, the reactions were mutagenesis, four to eight colonies were terminated during the log phase of the picked, DNA was prepared by miniprep- reaction. A titration of enzyme amount arations (from 3-ml cultures) and se- and the number of cycles were per- quenced using Sequenase 2.0 (U.S. Bio- formed each time a new enzyme prepa- chemical). Five sequencing primers ration was used. In general, our titra- distributed over the entire P/C gene tions indicated that using 0.5 unit of were used to determine sequence errors Vent polymerase (New England Biolabs) FIGURE 1 Amplification products of pcPC as a result of amplification. One primer in 50 gl of reaction yielded a good quan- with Vent polymerase at indicated number of was used to authenticate the mutagenesis. PCR cycles. Number of cycles are presented tity of PCR product at 15 cycles. Thus, on the top of the respective lanes. Mock (M) amplifications were carried out in a S0- RESULTS AND DISCUSSION presents the template plasmid linearized at a gl reaction mixture [S00 ~tM dNTPs, 10 unique HindlIl site. Amplified 6.7-kb product mM KC1, 20 mM Tris-HC1 (pH 8.8), 10 Amplification of Plasmid DNA is clearly seen in the 15-cycle lane, but only mM (NH4)2SO4, 2 mM MgSO4, 0.1% Tri- faint bands are detected at this size in 20- and ton X-100] with 25 pmoles of each phos- To determine the appropriate number of 25-cycle lanes. No distinct band is seen in the phorylated primer pair and 100 ng of cycles for PCR reactions, we amplified 30-cycle lane.
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BYRAPPA E1 AL.
M 1 234 5 6 choose any target site on the plasmid for whereas the later mutagenesis was car- mutagenesis. ried out using pgfPC. Appropriate primer pairs (Table 1) were used to amplify the entire recom- Site-specific Mutagenesis binant plasmid as described above. The 8.5- full-length DNA product was purified, 6.6- 5.4'- Using the procedure described in Mate- self-ligated, and transformed into bacte- 3.5- rials and Methods, we created site-spe- rial cells as described above (Fig. 2). cific mutants in the P and C reading Figure 3 schematically represents the frames of the bicistronic P/C gene of overall procedure of site-directed mu- Sendai virus. The P reading frame is 568 tagenesis used. For each mutagenesis ex- amino acids long and encodes the viral periment, four to eight colonies were polymerase associated phosphoprotein, grown in minicultures and plasmid DNA whereas the C reading frame is 204 was purified and sequenced by chain- amino acids long and encodes a non- terminating dideoxynucleotides using FIGURE 2 Amplification products of recombi- structural protein of unknown function. Sequenase. In both single-base mutagen- nant plasmids pcPC and pgfPC. Primer pairs Our aim was to mutagenize Ser-249 esis, all six clones for Pro-250-Ala and P120/P121 and P124/P125 were used to am- (UCC) and Pro-250 (CCA) of the P read- Leu-5-Ter were authentic mutants. In plify pcPC and pgfPC. (Lane M) The molecu- ing frame to Asp (GAC) and Ala (GCA), the double-base mutagenesis of Ser-249- lar weight markers in kb. (Lanes 1, 4) Linear- respectively, and Leu-5 (UUA) in the C Asp, six out of eight clones were authen- ized pcPC, and pgfPC, respectively. (Lanes 2, reading frame to a terminator codon tic mutants (Table 1). This mutagenesis 3) The amplification products of pcPC with P120/P121 and P124/P125, respectively. (UAA). The first two mutations were car- was further authenticated by restriction (Lanes 5,6) The amplification products of ried out using recombinant pcPC, enzyme digestion and expression of the pgfPC with P120/P121 and P124/P125, re- spectively.
amplifying longer templates (>40 kb) us- ing a mixture of thermostable DNA polymerases (]3,]4) indicates that mu- tagenesis of long templates will be pos- sible with optimized conditions and ap- propriate polymerases.
Design of Primers Mutagenic primers have been designed previously to carry the mismatch bases in the center of the primers flanked by complementary arms. (1-a's) However, we reasoned that for stable hybridiza- M C2 tion of a mutagenic primer to its tem- C1 C3 M C2 plate, a longer complementary arm with C1 C3 M C2
S' terminal mismatch sequences may be C1 C3 more suited for site-specific mutagenesis by PCR. Therefore, we designed 21-mer primers (P121 and P125) such that they carried 1 or 2 base mismatches at their 5' Tramtormation of E co# termini (Table 1). Alternatively, the 20- mer primer (P122) had a mismatch 6 nu- cleotides from the 5' end (Table 1). As all the primers worked efficiently in mu- tagenesis, we believe that mismatches at the 5' termini in mutagenic primers may I sek~;-~ Dn~ F~i~ant ~ [ be a better means of designing appropri- Ve~ IVUagene~ by Sequendr~ ate primers. In addition, because primer FIGURE 3 Schematic presentation of the protocol for the oligonucleotide-directed site-specific pairs with wide differences in their melt- point mutagenesis (left) and deletion mutagenisis (right) of a plasmid. M1 and C1 represent a ing temperatures and GC content primer pair with no gap between them except that the M primer contained a mismatch at the yielded appropriate products, we could S' end. C2 and C3 represent a primer pair designed to delete the filled-in region.
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PLASMID MUTAGENESIS
P and C proteins in CVl-transfected cific mutagenesis without phenotypic se- enzymatic amplification of DNA with a cells. In transfected cells the P and C lection. Methods Enzymol. 154: 367-382. thermostable DNA polymerase. Science proteins were expressed at the expected 2. Patwardhan, S., and K.C. Gupta. 1988. 239: 487-491. levels from mutants indicating that Vent Translation initiation potential of the S' proximal AUGs of the polycistronic P/C polymerase amplified the plasmid with mRNA of Sendal virus: A multipurpose high fidelity (data not shown). Restric- Received August 3, 1995; accepted in vector for site-specific mutagenesis. I. revised form October 19, 1995. tion digestions of the mutants revealed Biol. Chem. 263: 4907-4913. no detectable changes in the restriction 3. Higuchi, R., B. Krummel, and R.K. Saiki. fragment patterns, further indicating 1988. A general method of in vitro prep- the high fidelity amplification by Vent aration and specific mutagenesis of DNA polymerase (data not shown). To esti- fragments: Study of protein and DNA in- mate polymerase error rate during plas- teractions. Nucleic Acids Res. 16: 7351- mid amplification, we sequenced five 7367. different regions of the mutant P/C gene 4. Rashtachian, A., C.G. Thornton, and G. Heidecker. 1992. A novel method for site- spanning-1250 nucleotides. In total, we directed mutagenesis using PCR and sequenced >10,000 nucleotides from uracil glycosylase. PCR Methods ApDlic. several mutant plasmids and did not en- 2: 124-130. counter any error. This result indicated 5. Wong, F. and M. Komaromy. 1995. Site- that the Vent polymerase under our re- directed mutagenesis using thermDstal~le action conditions had a very low error enzymes. BioTechniques 18: 1034-1038. rate (<1 x l0 s) and amplified the plas- 6. Cariello, N.F., J.A. Swenberg, and T.R. mids with a high degree of fidelity. This Skopek. 1991. Fidelity of Thermococcus is consistent with a previous report. (6~ litoralis DNA polymerase (Vent TM) in However, these results underscore the PCR determined by denaturing gradient gel electrophoresis. Nucleic Acids Res. importance of reaction optimization; 19: 4193-4198. failing this, random undesirable muta- 7. Kong, H., R.B. Kucera, and W.E. Jack. tions may occur in the amplified mutant 1993. Characterization of a DNA poly- plasrnid. merase from hyperthermophile archaea As this procedure allowed efficient Thermococcus litoralis. J. Biol. Chem. mutagenesis of three entirely different 268: 1965-1975. plasmid vectors (two presented here and 8. Mattila, P., J. Korpela, T. Tenkanen, and one not presented), we believe that this K. Pitkanen. 1991. Fidelity of DNA syn- procedure will be universally applicable thesis by the Thermococcus litoralis DNA to generate mutants directly in plasmids polymerase--An extremely heat-stable enzyme with proof-reading activity. Nu- regardless of the plasmid origin. Follow- cleic Acids Res. 19: 4967-4973. ing essentially the identical protocol, we 9. Sambrook, J., T. Fritsch, and T. Maniatis. also obtained several deletion and inser- 1989. Molecular cloning: A laboratory man- tion mutants (not shown). The proce- ual. Cold Spring Harbor Laboratory Press, dure presented in this paper is more Cold Spring Harbor, New York. rapid and efficient at obtaining site-spe- 10. Cullen, B.R. 1986. Transactivation of hu- cific point and deletion mutants than man immunodeficiency virus occurs via previously described methods. (l'a'4's'ls~ a bimodal mechanism. Cell 46: 973-982. 11. Freier, S.M., R. Kierzek, J.A. Jaeger, N. Sug- imoto, M.H. Caruthers, T. Neilson, and D.H. Turner. 1986. Improved free-energy ACKNOWLEDGMENTS parameters for predictions of RNA duplex stability. Proc. Natl. Acad. Sci. 83: 9373- This research was supported by a re- 9377. search grant from the National Insti- 12. Imai, Y., Y. Matshushima, T. Sugimura, tutes of Health (AI30517). and M. Terada. 1991. A simple and rapid The publication costs of this article method for generating a deletion by PCR. were defrayed in part by payment of Nucleic Acids Res. 19- 2785. page charges. This article must therefore 13. Cheng, S., C. Fockler, W.M. Barnes, and be hereby marked "advertisement" in R. Higuchi. 1994. Effective amplification of long targets from cloned inserts and accordance with 18 USC section 1734 human genomic DNA. Proc. Natl. Acad. solely to indicate this fact. Sci. 91: 5695-5699. 14. Foord, O.S. and E.A. Rose. 1994. Long- distance PCR. PCR Methods Applic. 4: $149-$161. REFERENCES 15. Saiki, R.K., D.H. Gelfand, S. Stoffel, S.J. 1. Kunkel, T.A., J.D. Roberts, and R.A. Zak- Scharf, R. Higuchi, G.T. Horn, K.B. Mul- our. 1987. Rapid and efficient site-spe- lis, and H.A. Erlich. 1988. Primer-directed
GENOME RESEARCH ~ 407 Downloaded from genome.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press
A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase.
S Byrappa, D K Gavin and K C Gupta
Genome Res. 1995 5: 404-407 Access the most recent version at doi:10.1101/gr.5.4.404
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