PCR enhancement by organic solvents: Progress toward the development of chemical PCR

The polymerase chain reaction (PCR), an in vitro method for the amplification of DNA sequences, has rapidly become the central technique of modern molecular biology in the relatively short time since its discovery in 1985. Despite its widespread application, however, PCR amplification is often beset with the significant difficulties, in particular low yield of the target sequence and the nonspecific amplification of undesired side products. Although they can occur with a variety of different template types, these problems are especially severe with sequences of high GC content. GC rich sequences usually have greater melting temperatures (Tms), which reduces the efficiency of template denaturation, and are more prone to form stable single-stranded secondary structures, which impede the progression of the polymerase and result in the production of truncated products. In the present work we undertook a comprehensive investigation of the use of organic additives to enhance the yield and specificity of PCR amplification. Organic solvents have been found to significantly improve the yield and selectivity of a variety of simple enzymatic reactions, but their effects on complex biochemical reaction systems such as PCR, which include both DNA and protein, have not been examined in detail. The repertoire of PCR-enhancing compounds has historically remained limited to a small group of solvents because of the subtlety of the enhancement process. Drawing upon both precedent and inituition, we undertook a sweeping investigation of the abilities of four families of organic compounds---amides, sulfoxides, sulfones and diols---to improve PCR amplification. This extensive structure-activity study, encompassing over 40 compounds, uncovered several highly efficient and novel cosolvents that have become the state-of-the-art in PCR enhancement. We proceeded, then, to analyze the mechanistic modes of action of these compounds by examining their effects on various relevant parameters of the reaction. These investigations called for the development of highly sensitive fluorescence-based protocols for studying physico-chemical changes in the DNA template and polymerase under the influence of organic solvents. The mechanistic studies underscored the complexity of enhancement and suggested a reassessment of standard notions of solvent-induced macromolecular denaturation. Using current theoretical models of PCR amplification, augmented with modifications accounting for the presence of solvents, we found that it is possible in many cases to rationalize the observed trends in amplification enhancement based on our mechanistic data. This mechanistic understanding suggested, furthermore, that modification of conventional PCR cycling parameters might afford even greater improvements in the presence of organic solvents. Pursuing this idea, we found that certain organic solvents permit the use of cycling temperatures far outside the realm of standard PCR conditions, significantly increasing the versatility of the reaction. Within the framework of these expanded reaction conditions, the library of PCR-enhancing compounds discovered in the current work hold the promise of affording arbitrary selectivity in amplification in a manner analogous to the use of reagents to achieve selectivity in chemical reactions. The level of control inherent in chemical PCR may serve a critical role in extending the scope of the polymerase chain reaction to encompass a wide variety of emerging technologies that demand quantitative accuracy.