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

Abstract

PCR 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 co-solvents 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.

Evolution of Organic Solvent-Resistant DNA Polymerases

“…Other effective cosolvents include isobutyramide, 2-pyrrolidone, sulfolane, tetramethylene sulfoxide, and 1,3-propanediol, 10 with several of these being used as essential components in some of the most popular commercial PCR products� including isobutyramide in the widely used New England Biolabs Q5 high-fidelity polymerase mastermix and 1,3propanediol in multiple Toyobo PCR products. 17 While destabilizing dsDNA template and secondary structures in ssDNA template to improve amplification efficiency and reduce GC bias, these additives significantly reduce the thermostability and activity of the enzymes because DNA polymerases did not evolve in mixed aqueous-organic media. In our earlier work, we found that 1,4-butanediol is highly potent for the amplification of "difficult-to-amplify" templates 10 but deleteriously affects the polymerase itself (Figure S1).…”

“…PCR-enhancing additives, such as 1,4-butanediol, have been proven to be very effective, even making PCR amplification feasible for intractable templates, but negatively affect the polymerase. Other effective cosolvents include isobutyramide, 2-pyrrolidone, sulfolane, tetramethylene sulfoxide, and 1,3-propanediol, with several of these being used as essential components in some of the most popular commercial PCR productsincluding isobutyramide in the widely used New England Biolabs Q5 high-fidelity polymerase mastermix and 1,3-propanediol in multiple Toyobo PCR products . While destabilizing dsDNA template and secondary structures in ssDNA template to improve amplification efficiency and reduce GC bias, these additives significantly reduce the thermostability and activity of the enzymes because DNA polymerases did not evolve in mixed aqueous-organic media.…”

“…Due to high GC content, the amplification of the target DNA requires special PCR protocols, , organic solvent additives, , or addition of nucleotide analogs . Organic solvent additives, also known as “PCR-enhancing compounds”, have been used to improve GC-rich gene amplification or reduce GC bias in amplification without target modification and are components of some of the most commonly used PCR commercial products . Although PCR-enhancing organic solvents are very effective in improving amplification due to their favorable effects on dsDNA melting and single-stranded DNA (ssDNA) secondary structure alleviationthus complementing the effects of the increased temperatures used in PCRtheir application is limited by the fact that they deleteriously affect the polymerase stability and activity .…”

“…16 Organic solvent additives, also known as "PCR-enhancing compounds", have been used to improve GC-rich gene amplification or reduce GC bias in amplification without target modification and are components of some of the most commonly used PCR commercial products. 17 Although PCR-enhancing organic solvents are very effective in improving amplification due to their favorable effects on dsDNA melting and single-stranded DNA (ssDNA) secondary structure alleviation�thus complementing the effects of the increased temperatures used in PCR�their application is limited by the fact that they deleteriously affect the polymerase stability and activity. 10 Whereas polymerases used in PCR have evolved naturally to be thermostable, they have not evolved naturally to be solvent-tolerant, thus motivating the use of artificial directed evolution to improve them for such applications.…”

“…Moreover, WT activity is significantly reduced at 5% BD (TableS2). This analysis demonstrates that the polymerase characterization reported in this paper is of very broad and significant practical value, as it can be used to predict optimal conditions to enable the amplification of otherwise intractable GC-rich sequences.We also note in this regard that several commercial PCR reagents employ the closely related cosolvent 1,3-propanediol as a PCR enhancer,17,60,61 which has a smaller effective range than 1,4-butanediol. Effective range is an important criterion with respect to the scope of applications of a PCR enhancer because a small effective range can reduce the robustness of amplification to small errors in enhancer concentration or the presence of other inhibitory impurities.…”