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Abstract
Multichange ISOthermal(MISO) mutagenesis is a new technique allowing simultaneous introduction ofmultiple site-directed mutations into plasmid DNA by leveraging two existingideas: QuikChange-style primers and one-step isothermal (ISO) assembly.Inversely partnering pairs of QuikChange primers results in robust, exponentialamplification of linear fragments of DNA encoding mutagenic yet homologous ends.These products are amenable to ISO assembly, which efficiently assembles theminto a circular, mutagenized plasmid. Because the technique relies on ISOassembly, MISO mutagenesis is additionally amenable to other relevant DNAmodifications such as insertions and deletions. Here we provide a detaileddescription of the MISO mutagenesis concept and highlight its versatility byapplying it to three experiments currently intractable with standardsite-directed mutagenesis approaches. MISO mutagenesis has the potential tobecome widely used for site-directed mutagenesis.
Site-directed mutagenesis (SDM) is one of the most frequently used techniques inmolecular biology. The QuikChange reaction, developed by Stratagene (La Jolla, CA), isthe standard approach for introducing point mutations into plasmids. Its widespread useto make specific coding changes in proteins has driven fundamental discoveries in thefields of genetics, biology, and biochemistry. QuikChange uses reverse complementarymutational primers to replicate a parent plasmid, introducing mutation(s) at the site ofprimer binding. The DNA replication step is a linear cyclic amplification reaction inwhich a pfu polymerase copies the entire plasmid, stopping uponreaching the primer’s 5′ end; the newly synthesized DNA is nicked andcannot serve as a template in later cycles but rather anneals to form nickeddouble-stranded, mutagenized molecules. Enzymatic digestion of methylated template DNAproduced in E. coli using DpnI reduces the background of wild typeparental molecules in the reaction.
Since its invention, only modest improvements to the QuikChange reaction havebeen proposed,- and much inefficiency still exists in thissystem. First, while performed on a thermal cycler, DNA replication is linear notexponential. Second, strand displacement by the polymerase results in exponentialamplification of the plasmid and encodes a competing byproduct that cannot transformE. coli. Third, it is difficult to introduce mutations at more thanone location in any single reaction. Fourth, the size of the template is limitingbecause the method relies on replication of the entire plasmid. Fifth, because theentire plasmid must be copied, resequencing all nonselectable coding regions isobligatory. Sixth, background depletion by DpnI can be difficult because a large amountof DNA (50 ng) is recommended for the QuikChange reaction and hemimethylatedheteroduplex DNA is resistant to DpnI digestion., Seventh, becauseQuikChange primers are perfectly complementary, primer dimerization is highly favorable;long nick-bridging primers may minimize this and improve amplification. Finally, deletions and insertions largerthan a single codon are generally beyond the scope of the classic QuikChange reaction;however, modified QuikChange protocols attempt to address this shortcoming.
Herein we describe a simple and robust protocol for site directed mutagenesisthat overcomes all of the above challenges of the standard QuikChange reaction. We callour approach Multichange ISOthermal (MISO)mutagenesis since it is capable of introducing multiple DNA modifications in a singlereaction and incorporates a DNA assembly strategy named onestep isothermal (ISO)assembly. MISO mutagenesis isa completely different strategy from QuikChange; however, it still leverages the elegantdesign of QuikChange primers, which are reverse complementary sequences, usually 40 bpin length, that encode desired base changes centrally. In the simplest application ofMISO mutagenesis, two pairs of QuikChange primers are inversely partnered toexponentially amplify two linear, double-stranded PCR products (Figure 1a). The resulting PCR products encode the desiredmutations at each end and moreover share ~40 bp of terminal homology. Theone-step isothermal (ISO) reaction works by using a master mix of three enzymes toseamlessly assemble DNA pieces whose ends contain 30–40 base pairs ofoverlapping sequence (Figure 1b). Briefly, a5′ exonuclease chews back double-stranded DNA molecules to expose complementarysingle-stranded DNA overhangs. Homologous segments then specifically anneal. Next, apolymerase fills in the gapped molecules, and a ligase covalently seals nicks. Thus, PCRproducts with homologous ends, such as those generated through inverse partnering ofQuikChange primers, may be enzymatically joined in vitro using theone-step ISO assembly protocol to generate the desired mutagenized plasmid.
Overview of Multichange ISOthermal(MISO) mutagenesis. (a) QuikChange-style primer pairs (A, A′; B,B′) encode reverse complementary 40-nucleotide primers with a basesubstitution (star). Inverse partnering of primer pairs[A+B′] and[B+A′] in separate PCR reactions yieldsexponential amplification of two linear pieces of DNA with homologous ends.After template removal (DpnI digestion or gel purification), the mutagenizedplasmid is assembled using one-step isothermal assembly. (b) One-step isothermal assembly relieson the concerted action of three enzymes. A 5′ exonuclease chews backdouble-stranded DNA, exposing complementary single strands that anneal. Then apolymerase fills in the gaps, and a ligase seals the nick.
To demonstrate a robust capability for multi-site directed mutagenesis, we testedMISO mutagenesis with a set of 6 QuikChange primers encoding 8 base changes. Theseprimers were originally designed to incorporate eight lysine-to-arginine point mutationsinto a 6.5-kb plasmid using a combination of iterative QuikChange reactions and overlapextension PCR., We inversely partnered the six primer pairs(Figure 2a) to exponentially amplify sixdouble-stranded DNA fragments, ranging in size from 140 bp to 5.3 kb. The fragments weregel purified, subjected to one-step ISO assembly, and transformed into competentE. coli cells. As a control, one 140-bp fragment was omitted fromthe reaction, and the resulting assembly produced no colonies. Colony PCR reactions on96 individual transformants using two diagnostic primer pairs (Supplementary Figure 1a and b) revealedthat 92/96 clones had assembled correctly (Supplementary Figure 1c). Sequencing of 24correctly assembled constructs revealed that 100% contained all 8 desiredmutations. Thus, in a single round of experimentation we successfully generated alysine-free version of a protein of interest for assessment of post-translationalmodification status. These datasupport MISO mutagenesis as a tremendously improved strategy for multi-site directedmutagenesis.
Three applications of MISO mutagenesis. (a) Simultaneous introduction of eightpoint mutations into the mGOAT coding sequence. Six different pairs ofQuikChange-style primers were partnered (shown by colors) to generate six PCRproducts ranging in size from 140 bp to 5.3 kb and encoding eightlysine-to-arginine substitutions. One-step isothermal assembly reactionefficiently generated the desired lysine-free construct (see Supplementary Figure 1). (b)Introduction of a single point mutation into an existing 15.3-kb constructwithout vector segment amplification. QuikChange-style primers were partneredwith non-mutagenic primers complementary to plasmid ends to generate two PCRproducts (200 bp, 800 bp). Separately, pLD401 was digested with AscI and BstBI,and the large vector fragment was gel purified. The three DNA fragments, withhomologous ends, were subjected to one-step isothermal assembly to construct themutagenized plasmid. Only the region of the plasmid produced by PCR requiredconfirmatory resequencing. (c) Simultaneous introduction of a base substitution,deletion, and insertion into yeast shuttle vectors. One standard set ofQuikChange primers (starred) plus three other primer pairs were partnered (shownby color) to generate four overlapping PCR products. One-step ISO assemblyallowed deletion of two BsmBI sites from a noncoding region, recoding of oneBsaI site in the bla gene, and insertion of a BsaI-flanked RFPgene to generate a new cloning site. [Open gray arrows = codingsequence; stars = point mutations; scissors = unique restrictionenzyme sites; orange circles = undesirable restriction enzymerecognition sites; dashed lines = deleted region.]
Another limitation of QuikChange is the size of the template plasmid. As thereaction mandates replication of the entire construct, the upper limit for QuikChange is~7–10 kb. Further, many large plasmids carry a significant fraction ofcoding region, and any that cannot be functionally validated following QuikChange mustbe entirely resequenced to verify accuracy. Here we demonstrate additional versatilityof MISO mutagenesis to overcome these two issues by coupling MISO mutagenesis with atraditional restriction digestion. To introduce a single point mutation into a 15.3-kbplasmid of which ~9 kb encodes protein sequence (Figure 2b), we identifiedunique restriction enzymes sites flanking the desired base substitution by 200 bp and800 bp and designed primers to anneal beyond these boundaries. In individual PCRreactions, we inversely partnered these primers with two QuikChange-style primersencoding the mutation. Separately, the backbone was digested with the appropriaterestriction enzymes, and the three fragments were gel purified, thereby quicklygenerating three overlapping pieces of DNA amenable to one-step ISO assembly. Weconfirmed introduction of the mutation in five out of five unique transformants bysequencing. Further, one of these constructs was sequence verified at both overlappingjunctions. Here, not only did MISO mutagenesis allow efficient installation of themutation of interest, it also greatly reduced the amount of sequence validationrequired.
DNA modifications of biological relevance are not limited to base substitutions.Rather, the introduction of insertions and deletions into plasmids is often desirable,for instance, to generate fusion proteins, co-expression systems, or to delete proteindomains. To this end, we next used MISO mutagenesis to couple the introduction of apoint mutation with simultaneous deletion of a DNA segment and insertion of a 1-kbsequence into a series of yeast shuttle vectors (Figure2c). Briefly, we neededto recode a single BsaI site within the bla gene, remove two BsmBIsites from a noncoding region of the vector backbone, and construct a new cloning sitewith BsaI sites flanking a red fluorescent protein (RFP) to generate new host plasmidsamenable to Golden Gate assembly. Wedesigned QuikChange primers for BsaI recoding plus three additional pairs of primerswith overlapping overhangs (Figure 2c), which wereused to exponentially amplify four fragments with homologous ends. Gel purification ofall amplification fragments prior to one-step ISO assembly yielded ~99%red colonies, and 20 were miniprepped and the assembly tested by restriction digestswith BsaI and BsmBI (SupplementaryFigure 2). We discovered that 19/20 clones yielded the expected digestionpattern; the single incorrect clone derived from an assembly error in which oneRFP-flanking BsaI site was not intact (Supplementary Figure 2). This highlights that one-step assembly reactionsare prone to mis-assemblies and junctions must be sequence verified. Thus, afterfunctional verification by restriction digestion, we further sequenced the BsaI-RFPjunctions of 8 correctly assembled clones to verify elements that could not beinterrogated by digest; no undesired mutations were found. Taken together, theapplication of MISO mutagenesis presents a simple strategy for making many types ofbiologically relevant DNA modifications in a single round of experimentation.
MISO mutagenesis overcomes many specific technical problems associated withtraditional QuikChange mutagenesis. Since primers are inversely partnered in separatereactions, the problem of primer dimerization is circumvented. Further, MISO mutagenesisaffords exponential rather than linear amplification, thus allowing easy verification ofproduct generation by gel electrophoresis. Background is reduced almost completelythrough template removal by gel purification and/or DpnI digestion. Like QuikChange,however, MISO mutagenesis is limited by DNA sequences that are challenging to amplify byPCR, or may be toxic, unstable, or otherwise not tolerated in bacteria. The error rateassociated with oligonucleotide synthesis is another problem common to both approaches.Limitations specific to MISO mutagenesis derive largely from the ISO-assembly step.First, as demonstrated here, mis-assembly errors can occur during one-step ISO assembly,and it is likely that mis-assemblies will be exacerbated by repetitive or GC-richsequences in homologous regions. It is possible that implementation of alternativeenzymatic assembly strategies may overcome some types of assembly errors., Second, the introduction of mutations that are relatively closetogether (e.g., 50–80 bp) may be difficult to achieve using MISO mutagenesis, asthis would require exceedingly long complementary mutagenic primers to encode bothmutations or alternatively generating a very short PCR product. Thus, MISO mutagenesisis likely best applied when desired mutations are close enough to be encoded together onone primer or distant enough to generate a reasonably sized PCR product using twomutagenic primers. Finally, the number of pieces that can be put together by one-stepISO assembly defines the upper limit of DNA modifications introduced during a singleround of MISO mutagenesis. Of course, most of these limitations can be overcome byperforming MISO twice.
Overall, we believe that MISO mutagenesis is a versatile and efficient strategyfor making the most common types of DNA sequence modifications in plasmids. Thisapproach is accessible and cost-effective for all laboratories as it seamlesslyincorporates QuikChange-style primers, which are both familiar and ubiquitous inmolecular biology, and requires no expensive primer purification. Implementation of theone-step ISO assembly protocol is also straightforward as step-by-step instructions toprepare the three enzyme reaction mixture have been comprehensively detailed. Alternatively, kits with all therequired reagents preassembled are available from NEB. Thus, MISO mutagenesis representsan excellent solution for laboratories that infrequently perform SDM in plasmid DNA tothose where it is routine. Notably, single mutations can also be installed using MISOmutagenesis by inversely partnering QuikChange-style primers with a second pair ofnon-mutagenic primers or by employing the approach outlined in (Figure 2b). Indeed, we have used this approach to successfullysalvage failed QuikChange reactions (data not shown).
METHODS
Primer Design
Primers used in this study are listed in Supplementary Table 1. Formutagenic primers, 40 nucleotide exact reverse complementary sequences weredesigned with the base substitution placed centrally, as per the QuikChangemanual (Stratagene). In the case of insertions/deletions, the desired constructwas assembled in silico using the free plasmid editor ApE(http://biologylabs.utah.edu/jorgensen/wayned/ape/), keepingtrack of the junction locations. Primers were then designed to consist of twoparts: an annealing sequence (20–30 nt, Tm ≈ 55°C) and an ‘overhanging’ sequence to generate thehomologous region. In our hands, the minimal homologous region for ISO assemblyis ~30 bp, although 40 bp is recommended,,and we have succeeded in assembling fragments that overlap by as much as 200 bp.As an example, the primers used to generate Figure2c are diagramed in Supplementary Figure 3. In all cases, overlapping regions wereconfirmed to be unique to each assembly reaction.
PCR Amplification of DNA Fragments
Phusion Polymerase (NEB, F530L) was used to generate all PCR productsdescribed here, although any high fidelity polymerase is appropriate for use.PCR reactions were prepared as follows: 5–10 ng template DNA, 200μM concentration of each dNTP (Takara, 4030), 0.2μM concentration of each primer (Supp. Table 1), 1x Phusion HFbuffer, 0.02 U/μL Phusion DNA polymerase in a finalvolume of 50 μL. Applied Biosystem Veriti 96-WellThermal Cyclers were used for amplifications with an extension time of 30 s/kb.PCR products were either gel purified using the Zymoclean Gel DNA Recovery Kit(Zymo Research, D4002) as per the manufacturer’s instructions orpurified using the DNA Clean & Concentrator kit (Zymo Research, D4003)with or without DpnI treatment (NEB, R0176) for 1 h at 37 °C.
One-Step ISO Assembly
One-step ISO assembly reagents (5X ISO Buffer and Reaction Master Mix)are described in detail elsewhere. PCR products were combined in equimolar amounts in 5μL and mixed with 15 μL ofReaction Master Mix by gentle tapping. One-step isothermal assembly wasperformed at 50 °C in a preheated PCR block for 30 min, and 2μL of each assembly reaction was transformed into50 μL of competent DH5α E.coli cells.
Supplementary Material
Supplement
Acknowledgments
This work was supported in part by a National Science Foundation (grant MCB-1026068)and a DARPA contract (N66001-12-C-4020) to J.D.B.
ABBREVIATIONS
| MISO | multichange isothermal |
| ISO assembly | one-step isothermal assembly |
| PCR | polymerase chain reaction |
| DNA | DNA |
| nt | nucleotide |
| bp | base pair |
Footnotes
Author Contributions
L.A.M., Y.C., and M.T. are co-first authors of this work and wrote themanuscript. J.C., L.A.M., and M.T. created the figures. L.A.M., J.C., A.M.N.,Y.C., L.D., and M.T. contributed experimental data. The work was performed inthe lab of J.D.B.The authors declare no competing financial interest.
Supporting Information
Supplementary figures andtable. This material is available free of charge via the Internet athttp://pubs.acs.org.
References
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