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.

Highlights

  • Quantitative real-time PCR is becoming increasingly important for DNA genotyping and gene expression analysis

  • We have found that SYBR green I (SGI) inhibits amplification of medium-size genomic DNA fragments and that this inhibitory effect can be reduced by using a PCR mix, denoted here as mix IV, with modified salt composition [5]

  • We first tested whether the mix IV was optimal for Quantitative real-time PCR (qPCR) analysis with other DNA dyes

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Summary

Introduction

Quantitative 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. Used are sequence-specific probes which facilitate a highly sensitive detection of specific PCR products. These probes are difficult to prepare and are relatively expensive [1]. An alternative to the probe-based methods is the use of DNA-intercalating primers to the template during PCR [5]. This could account for some difficulties in amplifying certain DNA fragments, which are otherwise amplified in the absence of the dyes

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