1,2-propanediol-trehalose mixture as a potent quantitative real-time PCR enhancer

Abstract

BackgroundQuantitative real-time PCR (qPCR) is becoming increasingly important for DNA genotyping and gene expression analysis. For continuous monitoring of the production of PCR amplicons DNA-intercalating dyes are widely used. Recently, we have introduced a new qPCR mix which showed improved amplification of medium-size genomic DNA fragments in the presence of DNA dye SYBR green I (SGI). In this study we tested whether the new PCR mix is also suitable for other DNA dyes used for qPCR and whether it can be applied for amplification of DNA fragments which are difficult to amplify.ResultsWe found that several DNA dyes (SGI, SYTO-9, SYTO-13, SYTO-82, EvaGreen, LCGreen or ResoLight) exhibited optimum qPCR performance in buffers of different salt composition. Fidelity assays demonstrated that the observed differences were not caused by changes in Taq DNA polymerase induced mutation frequencies in PCR mixes of different salt composition or containing different DNA dyes. In search for a PCR mix compatible with all the DNA dyes, and suitable for efficient amplification of difficult-to-amplify DNA templates, such as those in whole blood, of medium size and/or GC-rich, we found excellent performance of a PCR mix supplemented with 1 M 1,2-propanediol and 0.2 M trehalose (PT enhancer). These two additives together decreased DNA melting temperature and efficiently neutralized PCR inhibitors present in blood samples. They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives. Furthermore, amplification in the presence of PT enhancer increased the robustness and performance of routinely used qPCRs with short amplicons.ConclusionsThe combined data indicate that PCR mixes supplemented with PT enhancer are suitable for DNA amplification in the presence of various DNA dyes and for a variety of templates which otherwise can be amplified with difficulty.

Negative regulatory roles of ORMDL3 in the FcεRI-triggered expression of proinflammatory mediators and chemotactic response in murine mast cells

“…Single-stranded cDNA was synthesized with M-MLV reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Real-time PCR amplifications of cDNAs were performed in 10 ll reaction volumes of qPCR mix containing 1 M 1,2-propanediol, 0.2 M trehalose and SYBR green 1 [34] in 384-well plates sealed with LightCycler 480 sealing foil and analyzed by LightCycler 480 (Roche Diagnostics). The following cycling conditions were used: 3 min at 95°C, followed by 50 cycles of 10 s at 95°C, 20 s at 60°C and 20 s at 72°C.…”

Role of Melt Curve Analysis in Interpretation of Nutrigenomics’ MicroRNA Expression Data

“…To optimize performance of the buffer, a PCR mix supplemented with two additives, 1M 1,2-propanediol and 0.2 M trehalose, were shown to decrease T m , efficiently neutralize PCR inhibitors, and increase the robustness and performance of qPCR with short amplicons (66). "uAnalyze SM " is another web-based tool, similar to uMELT, for analyzing high-resolution melting PCR products' data, in which recursive nearest neighbor thermodynamic calculations are used to predict a melt curve.…”

Massively parallel sequencing for early molecular diagnosis in Leber congenital amaurosis

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