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Technical Briefs |
1 Institute for Hormone and Fertility Research, Centre of Innovative Medicine, Falkenried 88, 20251 Hamburg, Germany
aauthor for correspondence: fax 49-40-42803-1699
Compatible solutes are a class of compounds that stabilize cells and cellular components exposed to extreme conditions. In bacterial systems, the uptake or synthesis of compatible solutes renders the cells and their enzymatic machinery more resistant to stress-inducing environmental conditions such as high osmolarity or high temperatures (1)(2). Compatible solutes comprise a heterogeneous group of compounds, covering amino acids and their derivatives (3), sugars (4), and more obscure compounds such as the pyrimidine derivative ectoine (5).
The compatible solute trehalose is a nonreducing disaccharide in which two D-glucose units are linked by an 
,-1,1-glycosidic bond. It is synthesized by a variety of eukaryotic organisms, conferring tolerance against desiccation, dehydration, heat, cold, and oxidation (6). The addition of trehalose increases the enzymatic activity of several euthermal enzymes used for cDNA synthesis or restriction digestion of DNA (7)(8). Trehalose also enhances the priming specificity in differential-display reverse transcription-PCR (9) through high-temperature priming and a thermoactivated reverse transcriptase.
PCR amplifications are frequently impaired by high GC content of the target sequence, leading to low yield and specificity of products, with no product at all in the worst cases. Locally high-temperature melting regions within the template can act as permanent termination sites (10). Several low-molecular-weight products have been identified that enhance the PCR of difficult templates, e.g., dimethyl sulfoxide (11) and other sulfoxides (12), formamide (13), nonionic detergents (14), and compounds belonging to the family of compatible solutes, such as betaine (15)(16)(17). The latter is present in most of the commercially available PCR-enhancing solutions (18).
Here we report the application of trehalose as a potent PCR enhancer for GC-rich templates. This compound avoids false negatives in PCR typing (16). In this study, we used trehalose in real-time, reverse transcription-PCR amplification of the mouse oxytocin receptor (mOT-R) transcript, which has a very high GC content. The identified molecular properties of this compound that lead to its PCR-enhancing ability are based on (a) lowering the melting temperature of DNA and (b) thermostabilization of the Taq polymerase.
Total RNA was prepared from 100 mg of mouse brain tissue by use of the RNeasyTM Mini Kit (Qiagen) according to the manufacturer’s protocol. cDNA synthesis was conducted according to the manufacturer’s protocol (SuperscriptTM; Invitrogen). Real-time PCRs were conducted with reagents as described (19) but adding various concentrations of D-(+)-trehalose (Sigma T-5251) in a total volume of 20 µL. Primers specific for mOT-R were mOT-R sense (5'-ttctacgggcccgacctgctgtgt-3') and mOT-R antisense (5'-ctgtgcggattttggccttggaga-3'; product size, 492 bp; positions 877-1368 of GenBank accession no. D86599), and those for the mouse ribosomal protein S27a were S27a sense (5'-ccaggataaggaaggaattcctcctg-3') and S27a antisense (5'-ccagcaccacattcatcagaagg-3'; product size, 296 bp; positions 117–413 of GenBank accession no. BC002108). Initial denaturation was for 3 min at 95 °C to activate the enzyme, after which 30–50 cycles of amplification were performed with denaturation at 95 °C for 30 s, annealing at primer-specific temperatures (mOT-R, 68 °C; S27a, 60 °C) for 10 s, and elongation at 72 °C for 30 s. Fluorescence data were acquired after 10 s at 80 °C.
After the cycling program, melting curve analysis was performed by cooling to 40 °C for 2 min and then increasing the temperature to 95 °C with a slope of 0.2 °C/s while measuring the fluorescence continuously. The melting peak was obtained by plotting the negative first derivative of fluorescence against temperature. The threshold cycle (crossing point), in which the fluorescence significantly exceeds background, was determined by the LightCyclerTM quantification software. Amplification efficiencies (slope) were calculated by a four-parameter sigmoidal fit (20) or, if not applicable, the "window-of-linearity" method (21). Fold differences were calculated by a mathematical model described elsewhere (22). All PCRs were conducted with six replicates. Statistical probabilities were determined by ANOVA and two-tailed t-statistics. All values presented are mean and SD.
The murine oxytocin mRNA (mOT-R) was chosen as a particular template possessing regions with a very high GC content and stable predicted secondary structures even at PCR elongation temperatures. A region within this template with a GC content of 85% was used as the experimental model. Trehalose enhances the amplification of mOT-R (Fig. 1
), with increasing amounts of trehalose lowering the crossing point. Even at high cycle numbers, product yield was low without trehalose, but it was increased significantly in a concentration-dependent fashion by trehalose up to a concentration of 0.4 mol/L. The gain in amplicon amount was 202-fold when we compared "no trehalose", with an efficiency of 1.46 (SD, 0.12), with 0.2 mol/L trehalose, which had an efficiency of 1.95 (SD, 0.19) and a cycle difference of 7.95 (SD, 0.52) cycles compared with no trehalose. At 0.6 mol/L, we observed a decrease in amplification efficiency, but this was based on the significant decrease in the primer melting temperature at this high concentration (data not shown). This effect can be circumvented by lowering the primer annealing temperature according to Table 1
.
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We consider the optimal concentration of trehalose to be 0.2 mol/L, a concentration at which the amplification of the GC-rich mOT-R significantly (P <0.0002) gains efficiency and primer annealing temperatures do not have to be adjusted. Betaine at 1 mol/L also significantly increased PCR efficiency (Fig. 1
). At 2 mol/L, we encountered a significant decrease in amplification efficiency, suggesting that betaine concentrations must be adjusted more carefully than do trehalose concentrations.
A melting program was run to verify the presence of only one PCR product. The resulting melting curves verified this by having only one melting peak, but additionally we observed a decrease in the melting point in correlation to the trehalose concentration for both templates (Table 1
). The DNA melting temperatures for mOT-R and S27a amplicons decreased by 0.5–1 °C for each 0.1 mol/L of trehalose. Betaine, already described as lowering the DNA melting temperature (23), also exhibited this characteristic on both amplicons, but only at concentrations >1 mol/L.
To elucidate the possible thermostabilizing effect of trehalose on the Taq polymerase, we incubated the enzyme at 95 °C for 30 and 90 min before using it in a S27a amplification. The incubation was conducted in the standard PCR buffer to ensure the same environment as in a typical amplification with template. When we used the thermally "stressed" Taq polymerase that had been incubated in the presence of trehalose (0.2 mol/L) in a subsequent PCR reaction, we observed a weak thermostabilizing effect after a preincubation of 30 min (Fig. 1
, inset). Amplification efficiencies were similar for Taq polymerase in the presence of trehalose (1.83; SD, 0.04) and in its absence (1.79; SD, 0.05). After 90 min of thermal preincubation in the absence of trehalose, a strong reduction in PCR efficiency was observed (1.57; SD, 0.16), which gave a relatively shallow amplification profile and low product yield after 35 cycles. Furthermore, the variation among the replicate samples was much higher. When the incubation was in the presence of 0.2 mol/L trehalose, the efficiency was lower than that for 30 min but significantly higher than at 90 min without trehalose (1.70; SD, 0.03). The variation among the replicates was also decreased compared with the latter conditions. Preincubation in the presence of 1.2 mol/L betaine also had a thermostabilizing effect compared with the control, but to a lesser degree than with trehalose, giving a lower PCR efficiency (1.67; SD, 0.08) and a higher crossing point.
For the exceptionally GC-rich oxytocin receptor, 35 cycles of amplification are needed to obtain a reasonable amount of PCR product, and the exponential phase of the amplification still exceeds this cycle number (24). The addition of increasing amounts of trehalose in the PCR cocktail strongly enhanced the PCR efficiency of the mOT-R, providing the desired amplicon amounts at much lower cycle numbers. Only at the highest concentration of trehalose (0.6 mol/L) did we observe a slight decrease in PCR efficiency, but this effect was based on the reduction of the primer annealing temperature, which must be adjusted accordingly at this concentration (data not shown). We and our coworkers have successfully used this compound with several templates with low amplification efficiency. For single PCR amplifications with GC-rich templates, it seems feasible to use high concentrations of trehalose to increase the PCR efficiency. If multiplex PCRs are run, or when a PCR master mixture is used with templates of varying GC content, a concentration of 0.2 mol/L, which does not affect the PCR efficiency of templates with a low GC content, is suggested.
The DNA destabilization by trehalose that we describe here might have two different effects that enhance PCR: (a) the overall lowering of the template melting temperature, which supports the single-stranded state in which the polymerase elongation can take place; and (b) the elimination of secondary structures that still persist in single-stranded state and can, in addition to a complete stop of the elongation step (10), lead to template switching of the Taq polymerase, mimicking splice variants because of small deletions (25).
Rendering several enzymes more resistant to thermal stress in the presence of trehalose, thereby maintaining or even increasing their enzymatic activity, has been described for MMLV reverse transcriptase, restriction enzymes, and low-temperature polymerases (7). Although trehalose renders Taq polymerase more stable when freezing PCR master mixtures (26), data supporting a high-temperature thermostabilizing effect of trehalose on thermostable polymerases used for PCR amplification have been missing. In the presence of trehalose, we observed a significant thermostabilizing effect, so that after 90 min of preincubation at 95 °C, the enzyme retained nearly all of its enzymatic activity compared with unstressed fresh enzyme. This also occurs, but to a lesser extent, with betaine, which has been described to enhance the PCR amplification of GC-rich templates (15)(17). This compound is routinely used at concentrations of 1–2 mol/L, which are much lower than the isostabilizing concentration of 5.2 mol/L (23). Therefore, it has been debated that the DNA-helix-destabilizing action of betaine alone cannot account solely for the PCR-enhancing effect (27)(28). The described ability of betaine to thermostabilize Taq polymerase might be the additional factor conferring the enhancing property.
In conclusion, trehalose is a compatible solute that greatly facilitates the PCR of GC-rich templates by reducing the DNA melting temperature and thermostabilizing the Taq polymerase. It thus could potentially be useful in the PCR amplification of difficult templates.
Acknowledgments
We thank D. Resuehr for fruitful discussions and the University Hospital of Hamburg (UKE) for providing excellent research facilities.
References
The following articles in journals at HighWire Press have cited this article:
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  G. M. Shaik, L. Draberova, P. Draber, M. Boubelik, and P. Draber Tetraalkylammonium derivatives as real-time PCR enhancers and stabilizers of the qPCR mixtures containing SYBR Green I Nucleic Acids Res., September 1, 2008; 36(15): e93 - e93. [Abstract] [Full Text] [PDF]
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