Trehalose Is a Potent PCR Enhancer: Lowering of DNA Melting Temperature and Thermal Stabilization of Taq Polymerase by the Disaccharide Trehalose,

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 α,α-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 …