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A Highly Efficient Procedure for Plasmids Using Vent DNA Polymerase

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A Highly Efficient Procedure for Plasmids Using Vent DNA Polymerase Downloaded from genome.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press 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, <e) we consistently had problems Recombinant Plasmids site-specific deletion, insertion, and in obtaining mutants using the cur- All DNA manipulations were carried out several bases mutagenesis facile and rently available PCR mutagenesis strate- following the standard protocols/9) The efficient. gies. Sendai virus P/C gene (-1.9 kb) <2) was Here, we present a simple, rapid, and cloned into pBC12/CMV/IL-2, ~1°~ into inexpensive PCR scheme to generate pcDNAI/amp (Invitrogen), and into mutants with a minimum number of pglfl ~2) yielding recombinant mole- Site-specific mutants I have played a steps. With only a single round of PCR, cules pBCPC (6.6 kb), pcPC (6.7 kb), and crucial role in defining structure-func- single-base mutants were obtained at a pgfPC (5.5 kb), respectively. Plasmid tion relationships of proteins and in de- frequency >90%. Our strategy is based DNA from minipreparations was used ciphering the significance of gene regu- on utilization of Vent polymerase, a for PCR amplifications as well as for latory elements. The recombinant DNA highly processive and high-fidelity ther- transfections in mammalian cells. technology for generating site-specific mophilic DNA polymerase from Thermo- mutants has developed rapidly in the coccus litoralis3 6-8) High processivity of last decade. Several novel strategies and the polymerase allows high-yield syn- Primers commercial kits have been developed to thesis of DNA fragments longer than 10 Primers (18-mer to 21-met) were custom generate oligonucleotide-directed site- kb with no requirements for additional synthesized by Integrated DNA Technol- specific mutants at a faster pace. In gen- constituents in the reaction mixture. As ogies, Inc. Primers were deprotected, eral, the various strategies fall into two most recombinant plasmids are <10 kb dried, resuspended in water, and quan- broad categories. In one, a mutagenic in size, Vent polymerase allows efficient titated by UV spectrometry. Each primer primer is annealed to a single-stranded amplification of an entire plasmid. The (1000 pmoles) was phosphorylated at its plasmid DNA template to prime synthe- high fidelity of Vent polymerase is pri- 5' end in a 40-btl reaction mixture [50 sis of the mutant strand. The mutant marily attributable to the potent inte- mM Tris-HC1 (pH 7.5), 10 mM MgC12, 10 strand can then be enriched by one of gral 3'-+5' exonuclease activity3 6'8) mM 2-mercaptoethanol, 10 nmoles of the several biochemical or biological However, high level of this activity ne- ATP, 15 units of polynucleotide kinase means to obtain the desired mu- cessitates an optimization of reaction (U.S. Biochemical)] at 37 ° for 30 min. At tants31'2) However, obtaining single- conditions with each new batch of the the end of incubation, 40 ~tl of water was 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.
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