Why do we use dmso in pcr

Those nuclear enzymes play vital roles in several cellular processes, such as replication, transcription, and recombination [ 5 — 8 ]. It is reported that some topoisomerases have been selected as the targets of several cancer chemotherapeutic agents [ 9 , 10 ]. Two types of topoisomerase are classified according to their mechanisms of catalysis: type I topoisomerases change the degree of supercoiling of DNA by causing single-strand breaks and religation, whereas type II topoisomerases cleave both DNA strands at the same time and pass another duplex strand through the break followed by sealing the double-strand break [ 11 , 12 ].

Dimethyl sulfoxide DMSO , on the other hand, is an important polar aprotic solvent that dissolves both polar and nonpolar compounds. As a result, it has been known as a good solvent for a wide range of organic compounds as well as water, which makes it possible to be used as a powerful tool in chemical and biological researches. Besides its ability to be a good solvent, DMSO has been known as a reagent to enhance the PCR amplification by inhibition of secondary structures in the DNA template or primers, especially in the synthesis of GC-rich gene fragments [ 16 , 17 ].

Moreover, DMSO has been involved in numerous modified molecular processes such as inhibition of cell proliferation [ 18 ], induction of cell differentiation [ 19 ], and apoptosis [ 20 ]. During the course of further mechanism studies, single-stranded or double-stranded oligonucleotides were employed as the inhibitors of the topoisomerases and the effects of the low concentrations of DMSO were also observed within those systems.

In addition, the evidence of topological structure changes caused by DMSO within negative supercoiled plasmid was examined by using atomic force microscopy AFM.

Shiga, Japan. All the buffers and solutions are prepared by the biological purity water. The resulting mixture was slowly cooled to room temperature. The obtained products were further analyzed using agarose electrophoresis 1. After that, 1. The obtained reaction mixture was analyzed as described above. All micas used in the current studies were modified on their surfaces with 3-aminopropyl triethoxysilane APS-micas following reported procedures [ 23 ].

Scan frequency was 1. All DNA sample determinations were carried out in air at room temperature. DMSO was reported to increase the single-stranded regions of negative supercoiled plasmid DNA, which are the crucial binding locations for the activity of type IA topoisomerases.

As shown in Figure 1 a , two bands were observed in each lane of the agarose gel electrophoresis: fast-moving bands lower bands were supercoiled plasmid while the slow-moving bands upper bands were DNA with relaxed conformation. We speculate that high order protein structures were disintegrated under the DMSO environment with higher concentrations and EcTopo I partially or fully loses its activities accordingly.

In addition, a control experiment was also conducted, in which the effect of DMSO on the plasmid substrate was tested. It has been reported that all type IB topoisomerases share a common fold around the active site region and a common catalytic mechanism [ 25 , 26 ]. It can be easily predicted that the reaction will not stop until there is no torsional strain to drive the swiveling and DNA is fully relaxed.

Different from EcTopo I, no enhancement of the relaxation efficiency can be observed Figure 2. In addition, CtTopo I are large proteins that contain multiple structural components which may be denatured easily even in a very low concentration of DMSO.

It has been reported in the past that eukaryotic topoisomerase I will lose its catalytic activity after it was preincubated with short ODNs.

This happens because the short ODNs can form covalent or noncovalent bonds with topoisomerases ODNs-enzyme complex , which will decrease the effective concentration of the topoisomerases in the reaction solution [ 28 ]. The same mechanism studies were also conducted during our investigations.

The result shown here corresponds with the established mechanism of EcTopo I, where type IA enzymes preferentially bind to the ssDNA region of the supercoiled plasmid [ 13 ]. Atomic force microscopy AFM has been known to be a powerful tool for determining certain subtle alternations in DNA topological features [ 23 , 30 , 31 ]. For the purpose of comparison, the pure negatively supercoiled plasmid was examined firstly. As shown in Figure 4 a , a compact supercoiled structure can be observed. It is known, on the other hand, that the entire topological structure of plasmid will be changed if there are some locally loose regions caused by the environment [ 17 ].

As shown in Figure 4 b , the topological molecular skeleton became loose comparing with the pure negatively supercoiled DNA molecules shown in Figure 4 a. We believe that the reason why the locally loose or single-stranded regions cannot be observed in Figure 4 b is that the areas are too small to be identified and they are beyond the resolution limitations of our AFM. In addition, most of the molecules bands are negatively supercoiled Lane 7 in Figure 1 a , which is contrary to the results shown in Figure 4 c.

This happens because the negatively supercoiled pBR DNA molecules are very sensitive to the concentration of DMSO and the condition in agarose gel running system is quite different from those DNA samples in the tubes. Therefore, the topological conformations of DNA molecules were changed in agarose gel, which was caused by the dilution of the concentration of DMSO in the gel. Since the sequence of pBR DNA contains an inverted repeated region, a cruciform structure can be formed within the negatively supercoiled pBR [ 32 , 33 ].

For the comparison purpose, negatively supercoiled pBR was first incubated with T7 endonuclease I in buffer solution that contains no DMSO for different reaction time. As shown in Figure 5 a , two new bands were observed.

The upper band is plasmid with nicked form and the lower band is linear DNA. The result shown here is consistent with the discussion in Figures 1 — 4. In addition, supercoil-induced formation of cruciform structure is greatly influenced by the salts and temperature [ 34 ].

The conditions used in our AFM studies do not favor the formation of cruciform structure and no such structure was observed Figure 4. Since the double helix is underwound in the negatively supercoiled plasmid, the single-stranded characters can be found. In the early stage of the reaction, an equilibrium situation occurs between the plasmid with negative supercoiling and DNA molecules with some single-stranded regions in a certain buffer and temperature condition. It has been established that type I enzymes alter linking number in steps of one, removing one supercoil i.

In Synthetic Biology, de novo synthesis of GC-rich constructs poses a major challenge because of secondary structure formation and mispriming. While there are many web-based tools for codon optimizing difficult regions, no method currently exists that allows for potentially phenotypically important sequence conservation.

Therefore, to overcome these limitations in researching GC-rich genes and their non-coding elements, we explored the use of DMSO and betaine in two conventional methods of assembly and amplification. Though we found no benefit in employing either DMSO or betaine during the assembly steps, both additives greatly improved target product specificity and yield during PCR amplification.

Of the methods tested, LCR assembly proved far superior to PCA, generating a much more stable template to amplify from. We further report that DMSO and betaine are highly compatible with all other reaction components of gene synthesis and do not require any additional protocol modifications.

Furthermore, we believe either additive will allow for the production of a wide variety of GC-rich gene constructs without the need for expensive and time-consuming sample extraction and purification prior to downstream application. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist. Since the de novo synthesis of the suppressor transfer RNA gene was first reported three decades ago [1] , our ability to engineer and assemble synthetic gene constructs has revolutionized the field of biomedicine [2] — [5]. Yet, despite our many achievements from assembling multi-kilobase plasmids to whole genomes [6] , [7] , de novo synthesis of GC-rich fragments remains a major obstacle namely because of secondary structure formation.

Sequences populated with G repeats produce complex inter and intrastrand folding due to increased hydrogen bonding with neighboring guanines at their N-7 ring positions [8]. In PCR, this phenomenon is marked by the appearance of shorter bands following gel electrophoresis.

These truncated versions of the target amplicon are primarily the consequence of arrest sites hairpins introduced into the template causing premature termination to polymerase extension [9]. In addition, mispriming and mis-annealing between template and compliment strands due to high melting temperature T m overlaps may contribute to incorrectly amplified gene constructs [10].

Because of these complications, GC-rich sequences are typically optimized by the researcher using web-based tools [11] — [14] that disrupt G repeats by choosing synonymous codons with lower T m s. However, there may be instances where nucleotide conservation is essential [15] — [18] particularly for non-coding regions where secondary structure functions to activate or repress transcriptional initiation [19]. While techniques are available to manage these difficult regions during PCR amplification of plasmid and genomic DNA [20] , [21] , to our knowledge no method for de novo synthesis of GC-rich templates has been clearly defined.

The closest application we found was GeneDesign [22] , which has the option to circumvent base rearrangement by adjusting the overlap between complimentary strands. As a cheap and effective approach to disrupting secondary structure formation and minimizing high T m ODN overlaps in de novo synthesis, we explored the use of the more popular and often referenced chemical agents, Dimethyl Sulfoxide DMSO [23] , [24] and betaine [25] , [26] during both the assembly and PCR amplification steps in conventional gene synthesis.

These isostabilizing agents facilitate strand separation of double helix DNA by altering its melting characteristics. For example, betaine, an amino acid analog with both positive and negative charges close to neutral pH, acts to equilibrate the differential T m between AT and GC base pairings; DMSO on the other hand, acts by disrupting inter and intrastrand re-annealing.

In this case, complimentary ODNs are denatured and annealed over several cycles for optimum strand alignment. A final round of PCR is then employed in both methods to amplify the target product using outside primers.

Though we only tested two genes, incorporation of either additive could aid in the construction of most GC-rich sequences. Protocol manipulation of standard conditions is also unnecessary due to the isostabilizing properties of these additives. Even without the need for nucleotide conservation, this application saves a great deal of end-user time not having to re-design and codon optimize ODNs prior to synthesis.

As such, the possibility of manually introducing sequence error is also limited; one mismatch, deletion or insertion could lead to a frame-shift or other gene lethality.

Furthermore, DMSO and betaine are very inexpensive, easily obtainable and highly compatible with other biological agents, which make them ideal for any gene synthesis assay.

Though this program has the option of calculating the optimum length of overlap given a target uniform T m , no such parameters were defined for either construct. After lyophilization, ODNs were resuspended and the optical density for each was measured at nm using a Spectramax Plus well plate reader. Samples were electrophoresed through a 1. DMSO IGF2R was chosen as a template to determine if the same additives, DMSO and betaine used to successfully amplify the fully formed gene fragment [25] could also be employed to aid in building and amplifying it from a pool of overlapping, single-stranded ODNs.

The rest of the gene fragment from bases to averages With respect to the hybridization map generated from Gene2Oligo [14] , T m s for the 20 bp overlaps average First, we tested if these additives had any effect on the assembly stage alone using the PCA method Fig.

Here, samples were ethanol precipitated to eliminate carry-over from assembly to the amplification step. A 1 kb DNA ladder in the outermost lanes marked at , and bp indicates the area of highest product band population. It was rationalized that tiling ODNs through ligation would better stabilize the template strands prior to their amplification. Similar to PCA, we introduced additives only during the ligation assembly step prior to ethanol precipitation. Neither DMSO nor betaine showed any marked influence on product formation using the stock polymerase mix for PCR when compared with the control samples Fig.

Only when additives were introduced during the amplification step did target yield and specificity greatly improve Fig. Growth curve assays were performed as in North et al. Briefly, a culture containing GFP-tagged wild-type and untagged mutant cells was treated with DMSO, and a ratio of growth was calculated for untagged cells in treated versus untreated samples, as compared to the GFP strain. All graphs display the mean and standard error of three independent cultures.

Three tests—regular t -test, Welch's test t -test modification assuming unequal variances and Wilcoxon Rank Sum Mann—Whitney test—were simultaneously applied to assess how possible violations of the assumptions underlying t -test homoscedasticity and normality affect statistical inference outcomes for the data.

Raw p -values for each test statistic were corrected for multiplicity of comparisons using Benjamini—Hochberg correction. P -values indicated on graphs are derived from regular t -tests, with Welch and Wilcoxon Rank Sum test results which are more robust but more conservative in terms of adjusted p -values usually in agreement with regular t -tests Table S1. To identify the biological attributes required for DMSO tolerance, enrichment analyses for the 40 sensitive strains was performed with FunSpec at a corrected p -value of 0.

Table 1. Table 2. Overrepresentation analyses suggested that subunits of COG, a protein complex that mediates fusion of transport vesicles to Golgi compartments, were required for DMSO tolerance. Growth curve assays also confirmed sensitivity of the individual COG deletions under non-competitive conditions Figure 1B.

Figure 1. Mutant strains were grown in competition with a GFP-expressing wild-type strain in the indicated DMSO concentrations and relative growth ratios treatment vs. The ratio means and standard errors are shown for three independent cultures. Growth curves for three independent cultures were obtained for the indicated strains and doses of DMSO.

Figure 2. Relative growth ratios were obtained for three independent cultures and analyzed as described in Materials and Methods. Growth curves were acquired from three independent cultures at the indicated doses. Figure 3. Various chromatin remodeling mutants are sensitive to DMSO.

For A—C,E , relative growth ratios were obtained and averaged for three independent cultures, while D displays average area under the curve data for growth curves acquired from three cultures. Figure 4. Relative growth assays were performed for three independent cultures. Figure 5. Growth curves for three independent cultures were obtained in the indicated doses of DMSO. The area under the curve AUC means and standard error are shown.

DMSO is a polar and aprotic solvent commonly utilized to solubilize chemicals during toxicological or pharmaceutical inquiries Santos et al. Compared to other solvents within its class such as sulfolane, N,N -dimethylformamide, N -methyl-pyrrolidinone, or N,N -dimethyl acetamide, DMSO exhibits relatively limited acute toxicity Tilstam, , thus affording it preferred status within these fields.

Despite its universality, DMSO's molecular mechanism s of action remain ambiguous, thus requiring investigations into the cellular processes and pathways it may perturb.

Here we conducted a genome-wide functional screen in the model eukaryote S. These results may indicate that DMSO's mechanism of toxicity in yeast is different from that exhibited in nematodes or human cells. However, if the toxic mechanism remains similar, it is feasible that compensatory cellular processes or genes are present in these mutants.

During the preparation of this manuscript, a report was published describing functional profiling of yeast mutants in DMSO Zhang et al. In this section, we discuss various aspects differentiating our investigation from Zhang et al. First, while these researchers assessed growth of individual yeast mutants via colony size on solid media, we performed functional profiling in pooled liquid cultures under competitive growth conditions.

Our analyses, in which DNA sequences unique to each strain are hybridized to a microarray after toxicant exposure, are able to discern small growth defects and can identify sensitive strains overlooked by other methods Table 4. However, the stringency of our DSSA may hinder identification of slow growing strains or those close to background levels.

Nevertheless, these data are extremely relevant to those conducting pooled growth assays, especially considering the increased popularity of automated screens and high-throughput multiplexed barcode sequencing to examine strain growth in DMSO-soluble toxicants or drugs Smith et al.

The contrasting choice of doses may also account for differences in the DMSO-sensitive strains identified by each screen. Finally, our overexpression data demonstrates that increased levels of Htz1p or Arp6p can rescue the growth of various deletion strains in DMSO Figure 5.

Table 4. A comparison between studies identifying yeast genes responsible for DMSO tolerance. Microarray analyses assessing the response of S. Consistent with these findings, Pommier et al. Z deposition into chromatin Meneghini et al. Z Morillo-Huesca et al.

Z at double-stranded DNA breaks Kalocsay et al. To separate effects of DMSO from a compound of interest, it is crucial for future yeast profiling studies to recognize that various deletion strains may fall out of pooled cultures during treatment with DMSO-solubilized drugs or toxicants.

Data gathered by our study can direct additional experimentation to decipher the cellular and molecular mechanisms of DMSO action. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Smith, Chris D. Vulpe is leader on Project 2]. The content is solely the responsibility of the authors and does not represent the official views of the funding agencies. Brandon D.