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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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Related in: MedlinePlus

Enhanced performance of PCR mix II-PT in routine qPCR amplifications. cDNA from mouse bone marrow-derived mast cells was prepared, diluted 1:105 (1), 1:104 (2), 1:103 (3) or 1:102 (4) and amplified in SGI-supplemented PCR mix II-PT or LC 480 SGI using primer sets for Actin, GAPDH, ORMDL1_Fr. a or ORMDL1_Fr. b. In controls (C'), cDNA was replaced by H2O. The samples were analyzed by qPCR followed by agarose gel electrophoresis and staining with ethidium bromide. Lane L, DNA standard ladder with sizes indicated in kbp on the left. Regression coefficients (R2) and efficiencies (E) were calculated from the plots of Cq values versus log copy numbers and are presented only for samples with the expected melting temperature at all template dilutions. Data are presented as means ± S.D. calculated from three independent experiments performed in triplicates.
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Figure 5: Enhanced performance of PCR mix II-PT in routine qPCR amplifications. cDNA from mouse bone marrow-derived mast cells was prepared, diluted 1:105 (1), 1:104 (2), 1:103 (3) or 1:102 (4) and amplified in SGI-supplemented PCR mix II-PT or LC 480 SGI using primer sets for Actin, GAPDH, ORMDL1_Fr. a or ORMDL1_Fr. b. In controls (C'), cDNA was replaced by H2O. The samples were analyzed by qPCR followed by agarose gel electrophoresis and staining with ethidium bromide. Lane L, DNA standard ladder with sizes indicated in kbp on the left. Regression coefficients (R2) and efficiencies (E) were calculated from the plots of Cq values versus log copy numbers and are presented only for samples with the expected melting temperature at all template dilutions. Data are presented as means ± S.D. calculated from three independent experiments performed in triplicates.

Mentions: PCR mix II-PT could be used with advantage not only for amplification of DNA fragments from crude blood samples that are difficult to amplify, but also for routine qPCR analysis of cDNA fragments without time-consuming adjustment of qPCR conditions for individual primer sets. Data presented in Figure 5A show that for amplification of 138 bp fragment of actin cDNA (58% GC) mix II-PT supplemented with SGI gave comparable regression coefficients and efficiencies as the routinely used LC 480 SGI. Similar results were obtained when GAPDH cDNA fragment was analyzed (Figure 5B; 52% GC, 69 bp). However, when low abundant cDNA fragments were amplified, such as ORMDL1_Fr.a (Figure 5C; 43% GC, 171 bp) or ORMDL1_Fr.b (Figure 5D; 47% GC, 205 bp), detectable amplification at all concentrations of template cDNA and reasonable regression coefficients and efficiencies were obtained only in mix II-PT-supplemented samples. Interestingly, addition of 1,2-propanediol and/or trehalose at various concentrations to LC 480 SGI containing chemically modified Taq DNA polymerase did not improve performance of this PCR mix, but instead had an inhibitory effect (data not shown).


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)

Enhanced performance of PCR mix II-PT in routine qPCR amplifications. cDNA from mouse bone marrow-derived mast cells was prepared, diluted 1:105 (1), 1:104 (2), 1:103 (3) or 1:102 (4) and amplified in SGI-supplemented PCR mix II-PT or LC 480 SGI using primer sets for Actin, GAPDH, ORMDL1_Fr. a or ORMDL1_Fr. b. In controls (C'), cDNA was replaced by H2O. The samples were analyzed by qPCR followed by agarose gel electrophoresis and staining with ethidium bromide. Lane L, DNA standard ladder with sizes indicated in kbp on the left. Regression coefficients (R2) and efficiencies (E) were calculated from the plots of Cq values versus log copy numbers and are presented only for samples with the expected melting temperature at all template dilutions. Data are presented as means ± S.D. calculated from three independent experiments performed in triplicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102612&req=5

Figure 5: Enhanced performance of PCR mix II-PT in routine qPCR amplifications. cDNA from mouse bone marrow-derived mast cells was prepared, diluted 1:105 (1), 1:104 (2), 1:103 (3) or 1:102 (4) and amplified in SGI-supplemented PCR mix II-PT or LC 480 SGI using primer sets for Actin, GAPDH, ORMDL1_Fr. a or ORMDL1_Fr. b. In controls (C'), cDNA was replaced by H2O. The samples were analyzed by qPCR followed by agarose gel electrophoresis and staining with ethidium bromide. Lane L, DNA standard ladder with sizes indicated in kbp on the left. Regression coefficients (R2) and efficiencies (E) were calculated from the plots of Cq values versus log copy numbers and are presented only for samples with the expected melting temperature at all template dilutions. Data are presented as means ± S.D. calculated from three independent experiments performed in triplicates.
Mentions: PCR mix II-PT could be used with advantage not only for amplification of DNA fragments from crude blood samples that are difficult to amplify, but also for routine qPCR analysis of cDNA fragments without time-consuming adjustment of qPCR conditions for individual primer sets. Data presented in Figure 5A show that for amplification of 138 bp fragment of actin cDNA (58% GC) mix II-PT supplemented with SGI gave comparable regression coefficients and efficiencies as the routinely used LC 480 SGI. Similar results were obtained when GAPDH cDNA fragment was analyzed (Figure 5B; 52% GC, 69 bp). However, when low abundant cDNA fragments were amplified, such as ORMDL1_Fr.a (Figure 5C; 43% GC, 171 bp) or ORMDL1_Fr.b (Figure 5D; 47% GC, 205 bp), detectable amplification at all concentrations of template cDNA and reasonable regression coefficients and efficiencies were obtained only in mix II-PT-supplemented samples. Interestingly, addition of 1,2-propanediol and/or trehalose at various concentrations to LC 480 SGI containing chemically modified Taq DNA polymerase did not improve performance of this PCR mix, but instead had an inhibitory effect (data not shown).

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
Related in: MedlinePlus