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

Horáková H, Polakovičová I, Shaik GM, Eitler J, Bugajev V, Dráberová L, Dráber P - BMC Biotechnol. (2011)

Bottom Line: We 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Signal Transduction, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic.

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusions: The 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.

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The effect of various DNA dyes and enhancers on ssDNA fluorescence and dsDNA melting temperature. (A) TNF-1 oligonucleotide (ssDNA, 45.5% GC; 1 μM final concentration) in PCR mix II (without dNTPs, Taq DNA polymerase and anti-Taq) was mixed with H2O (Control; Co) or enhancers [0.2 M trehalose (T; final concentration), 1 M 1,2-propanediol (P) or both 1 M 1,2-propanediol and 0.2 M trehalose (PT)] and various DNA dyes at final concentrations as indicated in Table 1. After heating at 95°C for 2 min the samples were cooled to 50°C and fluorescence was determined using Mastercycler ep realplex. (B) Oligonucleotide primer No 7, reverse (ssDNA; 72.2% GC; 1 μM final concentration) in PCR mix II was combined with various additives and DNA dyes, and fluorescence at 50°C was determined as in A. (C) Oligonucleotide mixture of TNF-1 and anti-TNF-1 (dsDNA, 45.5% GC; 1 μM final concentration) was prepared in mix II supplemented with various additives and DNA dyes. The samples were heated to 95°C for 2 min, then cooled to 30°C and temperature-dependent changes in fluorescence were obtained during heating from 30 to 95°C (0.2°C increments) in Mastercycler ep realplex. Melting temperatures were determined from the melting curves. (D) Oligonucleotide mixture of the primer No 7, reverse, and the anti-primer 7 (dsDNA, 72.2% GC; final concentration 1 μM) was combined in mix II with additives and DNA dyes and analyzed as in C. Means ± S.D. were calculated from 3 - 5 measurements.
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Figure 6: The effect of various DNA dyes and enhancers on ssDNA fluorescence and dsDNA melting temperature. (A) TNF-1 oligonucleotide (ssDNA, 45.5% GC; 1 μM final concentration) in PCR mix II (without dNTPs, Taq DNA polymerase and anti-Taq) was mixed with H2O (Control; Co) or enhancers [0.2 M trehalose (T; final concentration), 1 M 1,2-propanediol (P) or both 1 M 1,2-propanediol and 0.2 M trehalose (PT)] and various DNA dyes at final concentrations as indicated in Table 1. After heating at 95°C for 2 min the samples were cooled to 50°C and fluorescence was determined using Mastercycler ep realplex. (B) Oligonucleotide primer No 7, reverse (ssDNA; 72.2% GC; 1 μM final concentration) in PCR mix II was combined with various additives and DNA dyes, and fluorescence at 50°C was determined as in A. (C) Oligonucleotide mixture of TNF-1 and anti-TNF-1 (dsDNA, 45.5% GC; 1 μM final concentration) was prepared in mix II supplemented with various additives and DNA dyes. The samples were heated to 95°C for 2 min, then cooled to 30°C and temperature-dependent changes in fluorescence were obtained during heating from 30 to 95°C (0.2°C increments) in Mastercycler ep realplex. Melting temperatures were determined from the melting curves. (D) Oligonucleotide mixture of the primer No 7, reverse, and the anti-primer 7 (dsDNA, 72.2% GC; final concentration 1 μM) was combined in mix II with additives and DNA dyes and analyzed as in C. Means ± S.D. were calculated from 3 - 5 measurements.

Mentions: In our previous study we showed that SGI bound to ssDNA primers and suggested that the binding could at least in part contribute to the inhibitory effect of SGI in qPCR assays [5]. In further studies we evaluated whether trehalose and/or 1,2-propanediol could interfere with interaction of various DNA dyes with ssDNA, as reflected by changes in fluorescence signal. Data presented in Figure 6A indicate that interaction of various DNA dyes (at concentrations used for qPCR) with ssDNA oligonucleotide primer for tumor necrosis factor (TNF; 45.5% GC) induced fluorescence in the following order: SGI < SYTO-13 < LCGreen < SYTO-9 < EvaGreen < ResoLight. Addition of 0.2 M trehalose had little to no inhibitory effect on this fluorescence. In contrast, 1,2-propanediol significantly (P < 0.05; n = 3-5) decreased fluorescence in all DNA dyes used, except for SGI. Combination of both 1,2-propanediol and trehalose had a similar effect as 1,2-propanediol alone. When ssDNA primer No. 7, reverse (Table 4; 72.2% GC) was used, basal level of fluorescence was increased in all DNA dye-enhancer combinations and again trehalose had no significant effect on fluorescence intensity. In contrast to trehalose, 1,2-propanediol significantly (P < 0.05; n = 4) decreased fluorescence, except for SGI and ResoLight (Figure 6B).


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

Horáková H, Polakovičová I, Shaik GM, Eitler J, Bugajev V, Dráberová L, Dráber P - BMC Biotechnol. (2011)

The effect of various DNA dyes and enhancers on ssDNA fluorescence and dsDNA melting temperature. (A) TNF-1 oligonucleotide (ssDNA, 45.5% GC; 1 μM final concentration) in PCR mix II (without dNTPs, Taq DNA polymerase and anti-Taq) was mixed with H2O (Control; Co) or enhancers [0.2 M trehalose (T; final concentration), 1 M 1,2-propanediol (P) or both 1 M 1,2-propanediol and 0.2 M trehalose (PT)] and various DNA dyes at final concentrations as indicated in Table 1. After heating at 95°C for 2 min the samples were cooled to 50°C and fluorescence was determined using Mastercycler ep realplex. (B) Oligonucleotide primer No 7, reverse (ssDNA; 72.2% GC; 1 μM final concentration) in PCR mix II was combined with various additives and DNA dyes, and fluorescence at 50°C was determined as in A. (C) Oligonucleotide mixture of TNF-1 and anti-TNF-1 (dsDNA, 45.5% GC; 1 μM final concentration) was prepared in mix II supplemented with various additives and DNA dyes. The samples were heated to 95°C for 2 min, then cooled to 30°C and temperature-dependent changes in fluorescence were obtained during heating from 30 to 95°C (0.2°C increments) in Mastercycler ep realplex. Melting temperatures were determined from the melting curves. (D) Oligonucleotide mixture of the primer No 7, reverse, and the anti-primer 7 (dsDNA, 72.2% GC; final concentration 1 μM) was combined in mix II with additives and DNA dyes and analyzed as in C. Means ± S.D. were calculated from 3 - 5 measurements.
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Related In: Results  -  Collection

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Figure 6: The effect of various DNA dyes and enhancers on ssDNA fluorescence and dsDNA melting temperature. (A) TNF-1 oligonucleotide (ssDNA, 45.5% GC; 1 μM final concentration) in PCR mix II (without dNTPs, Taq DNA polymerase and anti-Taq) was mixed with H2O (Control; Co) or enhancers [0.2 M trehalose (T; final concentration), 1 M 1,2-propanediol (P) or both 1 M 1,2-propanediol and 0.2 M trehalose (PT)] and various DNA dyes at final concentrations as indicated in Table 1. After heating at 95°C for 2 min the samples were cooled to 50°C and fluorescence was determined using Mastercycler ep realplex. (B) Oligonucleotide primer No 7, reverse (ssDNA; 72.2% GC; 1 μM final concentration) in PCR mix II was combined with various additives and DNA dyes, and fluorescence at 50°C was determined as in A. (C) Oligonucleotide mixture of TNF-1 and anti-TNF-1 (dsDNA, 45.5% GC; 1 μM final concentration) was prepared in mix II supplemented with various additives and DNA dyes. The samples were heated to 95°C for 2 min, then cooled to 30°C and temperature-dependent changes in fluorescence were obtained during heating from 30 to 95°C (0.2°C increments) in Mastercycler ep realplex. Melting temperatures were determined from the melting curves. (D) Oligonucleotide mixture of the primer No 7, reverse, and the anti-primer 7 (dsDNA, 72.2% GC; final concentration 1 μM) was combined in mix II with additives and DNA dyes and analyzed as in C. Means ± S.D. were calculated from 3 - 5 measurements.
Mentions: In our previous study we showed that SGI bound to ssDNA primers and suggested that the binding could at least in part contribute to the inhibitory effect of SGI in qPCR assays [5]. In further studies we evaluated whether trehalose and/or 1,2-propanediol could interfere with interaction of various DNA dyes with ssDNA, as reflected by changes in fluorescence signal. Data presented in Figure 6A indicate that interaction of various DNA dyes (at concentrations used for qPCR) with ssDNA oligonucleotide primer for tumor necrosis factor (TNF; 45.5% GC) induced fluorescence in the following order: SGI < SYTO-13 < LCGreen < SYTO-9 < EvaGreen < ResoLight. Addition of 0.2 M trehalose had little to no inhibitory effect on this fluorescence. In contrast, 1,2-propanediol significantly (P < 0.05; n = 3-5) decreased fluorescence in all DNA dyes used, except for SGI. Combination of both 1,2-propanediol and trehalose had a similar effect as 1,2-propanediol alone. When ssDNA primer No. 7, reverse (Table 4; 72.2% GC) was used, basal level of fluorescence was increased in all DNA dye-enhancer combinations and again trehalose had no significant effect on fluorescence intensity. In contrast to trehalose, 1,2-propanediol significantly (P < 0.05; n = 4) decreased fluorescence, except for SGI and ResoLight (Figure 6B).

Bottom Line: We 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Signal Transduction, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic.

ABSTRACT

Background: 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. 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.

Results: We 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.

Conclusions: The 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.

Show MeSH