DNA amplification in mixed aqueous-organic media: chemical analysis of leading polymerase chain reaction compositions

Abstract

AbstractPCR amplification of GC-rich regions often leads to low yield and specificity. Addition of PCR-enhancing compounds is employed in order to overcome these obstacles. PCR-enhancing additives are low molecular polar organic compounds that are included as undisclosed co-solvents in commercial PCR buffers. In the interest of transparency and to permit further optimization by researchers of PCR compositions for challenging amplification problems, we studied eight PCR buffers by GC/MS to identify and quantify their co-solvents. Buffer specificity, both rich in water and salified substances, required a suitable sample preparation before injection into the GC/MS system. The aqueous phase of each buffer was replaced by an organic solvent to remove, by precipitation and filtration, salified substances which are detrimental to the GC/MS analysis. This approach has demonstrated the advantage of eliminating both water and salified substances without any loss of co-solvents. The sensitivity of the developed method was demonstrated as the main co-solvents were easily detected, identified and quantified. The methodology for identifying the co-solvents is mainly based on comparison of both library matching of acquired MS spectra with NIST library and experimental mass spectra obtained from authentic chemical standards. For the quantification of each co-solvent, deuterated Internal standards of similar structure to the cosolvents were used to correct the variable recovery caused by sample preparation, matrix effects, and ion source variability. The recovery ratio of the developed method was verified and found to be in the range 90-120 %. We then characterized the effects of specific organic co-solvents identified during PCR amplification -- using DNA melting, polymerase thermostability, polymerase activity and real-time PCR methods -- in order to elucidate their mechanism of action and to permit further optimization of their effects on amplification efficiency and specificity.