Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR)

Polymerase chain reaction (PCR)-related technologies are hampered mainly by two types of error: nonspecific amplification and DNA polymerase-generated mutations. Here, we report that both errors can be suppressed by the addition of a DNA mismatch-recognizing protein, MutS, from a thermophilic bacterium. Although it had been expected that MutS has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. On the basis of this finding, we propose that MutS binds a mismatched primer-template complex, thereby preventing the approach of DNA polymerase to the 3′ end of the primer. Our simple methodology improves the efficiency and accuracy of DNA amplification and should therefore benefit various PCR-based applications, ranging from basic biological research to applied medical science.

PCR is completed. By contrast, our method prevents amplification from mishybridized primers or mutated template sequences by simply adding a thermostable MutS protein to the reaction.

Results and Discussion
To investigate the error-suppressing capability of MutS, we constructed a system to monitor the amplification efficiency of an 80-base pair (bp) template DNA (Supplementary Figure S1A) after 20 cycles of amplification, the midpoint of the PCR amplification curve (Supplementary Figure S1B). First, we determined whether TthMutS [17] could suppress nonspecific amplification caused by mishybridization of primers ( Figure 1A). Two types of mismatches were investigated; mispaired bases, such as GT, and unpaired bases, such as single-base insertion/deletion loops [6,7]. As representatives of such mispaired and unpaired bases, we used primers containing a GT mismatch or an unpaired T, respectively. In the absence of TthMutS, the mismatched primers had no effect on the amplification efficiency in standard PCR. By contrast, amplification from both types of mismatched primers was specifically suppressed in the presence of TthMutS ( Figure 1B,C). High concentrations of TthMutS slightly reduced amplification from perfectly matched primers. This may be explained by the fact that TthMutS also binds nonspecifically to DNA; therefore, nonspecific binding to the template DNA at the extension step could interfere with the progression of DNA polymerases (as discussed later). As shown in Figure 1C, TthMutS suppressed amplification by LA Taq, which contains T. aquaticus DNA polymerase I [18], an A family DNA polymerase. We confirmed that TthMutS was also effective with members of the two other DNA polymerase families; KOD polymerase [19] (B family) ( Figure 1C, middle panel) and Aquifex aeolicus DnaE [20] (DNA polymerase III α subunit) (C family) ( Figure 1C, right panel), indicating that TthMutS-dependent error suppression is effective with a wide range of DNA polymerases.  We also examined the effect of TthMutS on the amplification of the 80-bp template DNA by using real-time PCR and monitoring the increase in fluorescence of SYBR Green I, which specifically stains double-stranded (ds) DNA ( Figure 2A). The threshold cycle for each amplification was determined and plotted against the concentration of TthMutS in the reaction ( Figure 2B). The results clearly showed that TthMutS suppressed amplification from the mismatched primers more effectively than from the perfectly matched primers. The change in fluorescence (∆Rn) was plotted against the cycle number; (B) The threshold cycle (C t ) was determined and plotted against the TthMutS concentration. Blue, red and purple circles indicate amplification by using perfectly matched, GT-mismatched and unpaired T-containing primers, respectively. All experiments were repeated three times. Bars indicate standard deviations.
As a control, we tested the A. aeolicus MutL C-terminal domain [21], which is heat stable at 95 °C, but lacks mismatch-recognition ability (Supplementary Figure S2). This protein had no effect on template amplification with the mismatch-containing primers, indicating that the error-suppressing activity of TthMutS on nonspecific amplification is due to mismatch recognition.
These results demonstrate that TthMutS suppresses nonspecific amplification during PCR. We propose a model in which TthMutS binds to the mismatched primer-template complex at the extension step, thereby blocking the binding of the DNA polymerase to the 3' end of the primer ( Figure 3A). If this model is correct, we would expect the error-suppressive effect of TthMutS to be reduced by increasing the distance between the mismatch-bound TthMutS and the 3' end of the primer. Indeed, we found this to be the case. As shown in Figure 3B, the effect of TthMutS was reduced in proportion to the number of nucleotides between the mismatch and 3' end of the primers, substantiating our hypothesis. It should be noted that we cannot exclude the possibility that TthMutS also prevents elongation to the 5' end of the template, generating incompletely extended DNA fragments, which leads to unstable hybridization between the primers and the template at the next cycle. We next tested the ability of MutS to inhibit amplification from GT-mismatched or T-unpaired templates ( Figure 4A). We found that mismatches in the template did not affect amplification efficiency in the absence of TthMutS, but the addition of TthMutS resulted in mismatch-specific suppression of amplification ( Figure 4B,C). The effect of TthMutS was observed for all three DNA polymerase family proteins ( Figure 1C), and as expected, amplification was not affected by the A. aeolicus MutL C-terminal domain, which lacks mismatch-recognition capability (Supplementary Figure S3). We also monitored amplification of the perfectly matched and mismatched 80-bp templates by real-time PCR to evaluate the effect of TthMutS on the threshold cycle ( Figure 4D). TthMutS specifically increased the threshold cycle for the amplifications from mismatched templates, which showed good concordance with the results of the standard PCR experiments. TthMutS suppressed amplification of templates containing one, two or three mismatches with equal efficiency ( Figure 5A,B), indicating that a single mismatch is sufficient for TthMutS-dependent suppression; a possible mechanism is illustrated in Figure 5C. The template mismatches are uncoupled at the denaturation step, but are regenerated at the annealing step, where they are recognized by TthMutS. Binding of TthMutS then interferes with the progression of DNA polymerase that accompanies strand displacement [16,22,23]. This mechanism is analogous to that previously proposed by Mitani et al. (2007) for isothermal amplification. Another possible mechanism for the suppression of amplification from mismatched templates is that TthMutS may stabilize the dsDNA during the denaturing step. The stabilized templates may then fail to denature and be unavailable for further amplification. However, we found contrasting results by examining thermal denaturation of dsDNA by a dissociation curve analysis in real-time PCR. In these experiments, the addition of TthMutS did not affect thermal denaturation of perfectly matched, GT-mismatched or unpaired T-containing 21-bp dsDNA (Supplementary Table S1). To determine whether TthMutS is effective in error suppression during PCR amplification of longer templates, we amplified three different sequences (423-, 594-and 1278-bp) from T. thermophilus genomic DNA using Takara LA Taq and perfectly matched primers ( Figure 6A-F). To allow mishybridization of primers, the annealing step was performed at 48 °C, which is a relatively low temperature (see Methods Section). Nonspecific amplification was readily detected under these PCR conditions and in the absence of TthMutS. However, the addition of TthMutS to the reactions significantly improved the amplification specificity ( Figure 6). Because TthMutS is not stable at temperatures above 82 °C (Supplementary Figure S4), we supplemented the reaction with additional TthMutS after completion of the fifteenth cycle of the 30-cycle program. Our results show that 1.1 μM TthMutS was sufficient to suppress nonspecific amplification. In the same experiments, we also tested the thermostable A. aeolicus DNA-binding protein MutL [24], which binds nonspecifically to DNA, but does not recognize base pair mismatches. However, we found that A. aeolicus MutL suppressed both specific and nonspecific amplifications (Supplementary Figure S5). Although A. aeolicus MutL has weak endonuclease activity, DNA degradation was not apparent under the PCR reaction conditions employed (Supplementary Figure S6). A. aeolicus MutL exhibits approximately 10-and 100-fold higher affinity for perfectly matched dsDNA than does TthMutS and A. aeolicus MutL C-terminal domain (CTD), respectively [21]. The strong suppression of specific amplification by A. aeolicus MutL (Supplementary Figure S5) could be due to its nonspecific DNA-binding activity, which is of much higher affinity than that of TthMutS (K d values are 0.15 μM and 2 μM, respectively). This explains why A. aeolicus MutL showed much stronger suppression of specific amplification than TthMutS.
These results indicate that mismatch recognition is essential for the selective suppression of nonspecific amplification by TthMutS.  Figure 7A,B). We observed that TthMutS effectively suppressed nonspecific amplification at each cycle analyzed. To quantify the effect of TthMutS, we introduced the specificity factor, which is the ratio between the rate of suppression of nonspecific and specific amplification (see "Online Methods" for details). For the PCR cycle numbers tested with the 423-bp sequence, the estimated specificity factors of 1.1 μM TthMutS were between 10 and 20, as shown in Figure 7C. These estimates suggest that amplification was ~10-20-fold more specific in the presence of TthMutS than in its absence. Having demonstrated the ability of TthMutS to suppress nonspecific PCR amplification, we next examined its effect on DNA polymerase-generated mutations. For this, we amplified the 423-bp genomic fragment and cloned and sequenced the PCR products. To facilitate detection of a TthMutS effect, PCR was performed with LA Taq under error-prone conditions in the presence of 200 μM MnCl 2 . Our sequencing analysis showed that addition of 1.1 μM TthMutS reduced the frequency of polymerase-generated mutations by approximately two-thirds ( Figure 8A). Because MutS has weak ATPase and dATPase activities, we cannot exclude the possibility that the addition of TthMutS perturbs the dNTP balance, leading to misincorporation by DNA polymerases. To test this, we added equal concentrations of dATP and adenylyl imidodiphosphate (AMPPNP), which inhibits the ATP/dATPase activity of TthMutS. The addition of AMPPNP had no effect on the reduction of polymerase-generated mutations by TthMutS ( Figure 8B). This result suggests that the dATPase activity of TthMutS is weak and has little or no effect on the balance of dNTPs. A similar reduction in the polymerase-generated mutation rate was obtained when the experiment was performed in the absence of MnCl 2 ( Figure 8C). Because TthMutS is not stable above ~85 °C, we sought to improve the method by preparing MutS from the hyperthermophilic bacterium A. aeolicus, which can grow at temperatures of up to 95 °C [25]. A. aeolicus MutS (AaeMutS) was successfully overexpressed, purified and shown to be stable at 95 °C (Supplementary Figure S7). Of note, AaeMutS was able to suppress error-containing amplification of an 80-bp oligonucleotide ( Figure 9A,B) and a genomic DNA template ( Figure 9C,D). AaeMutS at a final concentration of 0.2-0.4 μM was sufficient to obtain mismatch-specific suppression. Importantly, AaeMutS exhibited a strong suppressive effect without the need to supplement the reaction with an additional protein at the end of the fifteenth cycle, as we had observed for TthMutS ( Figure 9E,F).
The PCR cycle-dependence of AaeMutS activity was tested by amplification of the 423-bp sequence over 20-40 cycles in the presence or absence of 0.4 μM AaeMutS ( Figure 10A,B). The specificity factors for 0.4 μM AaeMutS under these conditions were between 10 and 30 ( Figure 10C), which was very similar to those of TthMutS.  Figure 6A was performed using AaeMutS. The amounts of the products were normalized by that from perfectly-matched template and primers at 0 μM AaeMutS; (D) Quantification of the relative amounts of nonspecific (red) and desired (blue) amplification products in (C). The amounts of the products were normalized by the amount of total products at 0 μM AaeMutS; (E) The ttha1806 gene was amplified in the presence of 0.3 μM TthMutS or AaeMutS. At the end of the fifteenth cycle, 0 or 0.3 μM TthMutS or AaeMutS was added to each reaction tube. Control amplification in the absence of TthMutS or AaeMutS is indicated as "C"; (F) Quantification of the amplification products from (E). The amounts of the products were normalized by the amount of total products at 0 μM AaeMutS. Sequencing of PCR products showed that 0.3 μM AaeMutS decreased the number of polymerase-generated mutations to approximately 30% of the control level ( Figure 11A,B). From these results, we conclude that addition of AaeMutS is a convenient and useful method for suppressing both nonspecific amplification and polymerase-generated errors in PCR.  Figure 8A was performed using 0.3 μM AaeMutS; (B) The same experiment as in Figure 8C was performed using AaeMutS.
The major novel finding in this study is that MutS can suppress nonspecific amplification; that is, amplification from mismatched primers. Although we expected that MutS would be able to suppress amplification from a mismatched template, our results clearly demonstrate that MutS suppresses amplification from both mismatched templates and primers. Furthermore, by preparing MutS from the extremely thermophilic bacterium A. aeolicus, we also showed that the error-reducing effects of MutS can be applied to standard PCR conditions.

Overexpression and Purification of Proteins
The A. aeolicus mutS gene was amplified by PCR using A. aeolicus genomic DNA as the template. The forward and reverse primers were 5'-CCATGGAGAAATCTGAGAAAGAGCTCAC-3' and 5'-AGATCTTTATTAAGCTCCGGACTCCTTTTT-3', respectively, which contained NcoI and BglII sites, respectively (underlined). The amplified fragment was ligated into the NcoI and BamHI sites of pET-HisTEV [26] to yield a pET-HisTEV/A. aeolicus mutS plasmid. Sequence analysis revealed that the construction was error free.
E. coli BL21(DE3) (Novagen, Madison, WI, USA) was transformed with pET-HisTEV/A. aeolicus mutS and cultured at 37 °C in 1.5 L of YT medium [0.8% (w/v) tryptone, 0.5% (w/v) yeast extract and 0.5% (w/v) NaCl] containing 50 μg/mL ampicillin. When the culture density reached 4 × 10 8 cells/mL, isopropyl β-D-thiogalactopyranoside was added to a final concentration of 100 μM to induce expression. The cells were grown at 37 °C for a further 4 h, harvested by centrifugation and lysed by sonication in buffer I (20 mM Tris-HCl and 50 mM NaCl, pH 7.8). The sonicate was heated to 70 °C for 10 min, centrifuged at 48,000× g for 20 min, and the supernatant was then loaded onto a Talon resin column (40 mL; Clontech, Palo Alto, CA, USA) equilibrated with buffer I. The column was washed with 300 mL of buffer I and then eluted with buffer I containing 150 mM imidazole. The fractions containing AaeMutS were detected by sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) and collected, and (NH 4 ) 2 SO 4 was added to the fraction to a final concentration of 1 M. The solution was loaded onto a Toyopearl-Phenyl column (Tosoh, Tokyo, Japan) equilibrated with buffer I containing 1 M (NH 4 ) 2 SO 4 . The column was washed with 100 mL of buffer I containing 1 M (NH 4 ) 2 SO 4 and then eluted with a 300 mL gradient of 1 to 0 M (NH 4 ) 2 SO 4 in buffer I. The fractions containing AaeMutS were detected by SDS-PAGE and concentrated with a Vivaspin concentrator (Vivascience, Göttingen, Germany). The protein solution was dialyzed against 20 mM Tris-HCl and 50 mM NaCl (pH 7.8).
The A. aeolicus dnaE gene, which encodes a DNA polymerase III α subunit, was amplified by PCR using A. aeolicus genomic DNA as the template. The forward and reverse primers used were 5'-ACATATGAGTAAGGATTTCGTCCACCTTCA-3' and 5'-AGGATCCTTATTAAATTATGACC TTCACTCCCAG-3', respectively, which contained NdeI and BamHI sites, respectively (underlined). The fragment was ligated into the NdeI and BamHI sites of pET-HisTEV to generate a pET-HisTEV/A. aeolicus dnaE plasmid. Sequence analysis revealed that the construction was error free.
E. coli Rosetta 2(DE3) (Novagen) was transformed with pET-HisTEV/A. aeolicus dnaE. The cells were cultured and harvested using the same procedure as described above for A. aeolicus mutS overexpression. The product of A. aeolicus dnaE, AaeDnaE, was purified using the same procedure as described for AaeMutS.
TthMutS and the C-terminal domain of A. aeolicus MutL were overexpressed and purified as previously described [17,21].

Circular Dichroism Spectrometry
Circular dichroism measurements were carried out with a Jasco spectropolarimeter (J-720W; Jasco, Tokyo, Japan). All measurements were performed using a 0.1-cm cell at 25 °C. The residue molar ellipticity [θ] was defined as 100θ obs /lc, where θ obs was the observed ellipticity, l the length of the light path in centimeters and c the residue molar concentration of each protein. The measurements were performed in a solution consisting of 50 mM potassium phosphate (pH 7.0), 20 mM NaCl and 5 μM AaeMutS or TthMutS.

PCR Using Genomic DNA Template
The 5'-terminal 423-bp region of ttha1806 was amplified using the primers 5'-GAGACCA CCCGTAGGCGGCT-3' and 5'-CTTAAGGGGCCTCGCGCTCT-3'; a 594-bp region of tthb071 was amplified using 5'-CGTCAGGCTGGCCTTCCCCCTTTCC-3' and 5'-TTCCAGTGGCGGTCGTA GACCCCGTC-3'; a 1278-bp region of ttha1548 was amplified using 5'-GAGGAGGTGCTCTA CGTGGGCAAGGCC-3' and 5'-GGGAAGGTCCTTGAGGCTTCCCGTGTAGC-3'; and a 1515-bp region of ttha1645 was amplified using 5'-ATATCATATGCGTGACGTCCTCGAGGTCC-3' and 5'-ATATAGATCTTTATTACTCGAGCCTCTCCAGAAGGGCCTC-3'. The reactions were performed in 1× Takara GC I buffer (Takara) containing 0.06 units/μL LA Taq (Takara); 5 ng/μL T. thermophilus HB8 genomic DNA; 400 nM primers; 100 μM CoCl 2 ; 400 μM dATP, dTTP, dCTP and dGTP and various concentrations of TthMutS or AaeMutS. Thirty PCR cycles were run using an Astec OC707 thermal cycler. Conditions for amplification of ttha1806 and ttha1645 were as follows: denaturation step, 95 °C for 1 min; annealing, 58 °C for 1 min; and extension, 70 °C for 2 min. Amplification conditions for tthb071 and ttha1548 were as follows: denaturation step, 95 °C for 1 min; annealing, 50 °C for 1 min; and extension, 70 °C for 2 min. For reactions containing TthMutS, a second aliquot of TthMutS solution (equal to the amount in the initial reaction mixture) was added at the end of the fifteenth cycle. At the end of the program, 10 μL of reaction solution was mixed with 1 μL of loading buffer [50% (v/v) glycerol, 0.9% (w/v) SDS and 0.05% (w/v) bromophenol blue] and electrophoresed on a 1.5% agarose gel in 1× TBE buffer. The gel was stained with ethidium bromide, and the products were visualized under ultraviolet light at 254 nm. DNA products were quantified using ImageJ software. The "specificity factor" was defined as the ratio between the rate of suppression of nonspecific amplification and the rate of specific amplification and obtained using the following equation: Specificity factor at n μM MutS = A N0 A Sn /A Nn A S0 , where A N0 , A Sn , A Nn and A S0 are the amounts of nonspecific amplification at 0 μM MutS, specific amplification at n μM MutS, nonspecific amplification at n μM MutS and specific amplification at 0 μM MutS, respectively.
For the experiments examining polymerase-induced mutations, PCR reactions were performed using ttha1806 and ttha1548 templates in the presence of 0 or 200 μM MnCl 2 . The reaction products were ligated into the pT7Blue vector (Novagen, Madison, WI, USA) by using the TA cloning technique and sequenced using a BigDye terminator version 3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA).

Conclusions
Our new methodology offers a significant improvement over current PCR-related technologies. The suppression of nonspecific amplification will be important for a wide range of PCR-based technologies, including the detection of viral and bacterial infections and identification of SNPs. The observed reduction in polymerase-generated mutations (~65%) is also significant. For example, cloning a gene (typically 1000 bp) with a DNA polymerase that has low fidelity, but high elongation efficiency generally produces more than 1 mutation per 1000 bp ( Figures 8C and 11B). In such cases, the addition of MutS offers an attractive option to enable error-free recovery of the gene.
A thermostable RecA protein has previously been used to suppress nonspecific amplification during PCR, although this protein was unable to suppress polymerase-generated errors [28,29]. RecA promotes the correct hybridization of primers and template sequences, thereby reducing the frequency of mishybridizations. Thus, the mechanism by which MutS suppresses nonspecific amplification is quite different from that of RecA, suggesting that the combined use of MutS and RecA may allow even greater accuracy in PCR-based applications. Nucleic acid analogs, such as locked nucleic acids, peptide nucleic acids and morpholino oligomers have also been used as PCR probes to sustain the specificity of amplification [30]. The MutS-based technique described here could also be used in combination with such nucleic acid analogs. Further studies will be required to determine whether MutS can recognize a mismatch comprising a natural base and an analog.