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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.

Show MeSH
Amplification of medium-size GC-rich DNA fragment using whole blood as a source of the template. 806 bp fragment of human Q8N1R6 gene (73.3% GC) in heparinized human blood (2% final concentration) was amplified in PCR mix II containing various concentrations of trehalose (0.1 - 0.3 M) and 1,2-propanediol (0.5 - 1.0 M). Position of the specific product (Fr. 8) is shown by an arrow. A typical result of three independently performed experiments is shown.
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Figure 2: Amplification of medium-size GC-rich DNA fragment using whole blood as a source of the template. 806 bp fragment of human Q8N1R6 gene (73.3% GC) in heparinized human blood (2% final concentration) was amplified in PCR mix II containing various concentrations of trehalose (0.1 - 0.3 M) and 1,2-propanediol (0.5 - 1.0 M). Position of the specific product (Fr. 8) is shown by an arrow. A typical result of three independently performed experiments is shown.

Mentions: In an attempt to develop a universal PCR master mix compatible with all the DNA dyes and suitable for amplification of DNA templates that cannot be readily amplified due to dye interference, presence of inhibitory substances and/or secondary structure formation, we tested several additives combined with mixes I - IV and various DNA dyes. As a template we used GC-rich DNA fragment of Q8N1R6 gene (Table 4; 806 bp, 73.3% GC) in human heparinized blood which escaped detection under standard conditions using various commercial PCR master mixes such as iQ™ SYBR Green Supermix and LightCycler 480 SYBR Green I Master (LC 480 SGI). In pilot experiments various mixes were combined with several additives and/or procedures, which have been reported to allow amplification of GC-rich fragments and/or neutralize PCR inhibitory components present in the blood (hemoglobin, lactoferrin and immunoglobulin G [16,17]). These included 0.1 - 0.5 M trehalose (final concentration) [18], 5 - 15% dimethyl sulfoxide (DMSO) [19,20], 0.5 - 2.5 M N,N,N-trimethylglycine monohydrate (betaine) [21,22], combinations of 5 - 15% DMSO and 2.2 M betaine [23], 5 - 25 mM tetrapropylammonium chloride [5], 0.5 - 1.5 M 1,2-propanediol, 0.5 - 1.5 M ethyleneglycol [24], 50 - 150 μM 7-deaza-2'-deoxyguanosine 5'-triphosphate [25,26], PCR-enhancing coctail containing 0.3 M D-(+)-trehalose, 0.24 M L-carnitine, and 0.4% Nonidet P-40 (TCN) [27] and antibody-mediated hot start PCR [5] combined with "touchdown" procedure [28,29]. Yet, none of these additives and/or procedures improved PCR to get specific signal determined by agarose gel electrophoresis (data not shown). Interestingly, specific amplicons were observed in PCR mix II supplemented with both 1 M 1,2-propanediol and 0.2 M trehalose (mix II-PT); higher or lower concentrations of these two additives resulted in inhibition of production of the specific amplicons and/or enhanced formation of nonspecific DNA fragments (Figure 2).


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)

Amplification of medium-size GC-rich DNA fragment using whole blood as a source of the template. 806 bp fragment of human Q8N1R6 gene (73.3% GC) in heparinized human blood (2% final concentration) was amplified in PCR mix II containing various concentrations of trehalose (0.1 - 0.3 M) and 1,2-propanediol (0.5 - 1.0 M). Position of the specific product (Fr. 8) is shown by an arrow. A typical result of three independently performed experiments is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Amplification of medium-size GC-rich DNA fragment using whole blood as a source of the template. 806 bp fragment of human Q8N1R6 gene (73.3% GC) in heparinized human blood (2% final concentration) was amplified in PCR mix II containing various concentrations of trehalose (0.1 - 0.3 M) and 1,2-propanediol (0.5 - 1.0 M). Position of the specific product (Fr. 8) is shown by an arrow. A typical result of three independently performed experiments is shown.
Mentions: In an attempt to develop a universal PCR master mix compatible with all the DNA dyes and suitable for amplification of DNA templates that cannot be readily amplified due to dye interference, presence of inhibitory substances and/or secondary structure formation, we tested several additives combined with mixes I - IV and various DNA dyes. As a template we used GC-rich DNA fragment of Q8N1R6 gene (Table 4; 806 bp, 73.3% GC) in human heparinized blood which escaped detection under standard conditions using various commercial PCR master mixes such as iQ™ SYBR Green Supermix and LightCycler 480 SYBR Green I Master (LC 480 SGI). In pilot experiments various mixes were combined with several additives and/or procedures, which have been reported to allow amplification of GC-rich fragments and/or neutralize PCR inhibitory components present in the blood (hemoglobin, lactoferrin and immunoglobulin G [16,17]). These included 0.1 - 0.5 M trehalose (final concentration) [18], 5 - 15% dimethyl sulfoxide (DMSO) [19,20], 0.5 - 2.5 M N,N,N-trimethylglycine monohydrate (betaine) [21,22], combinations of 5 - 15% DMSO and 2.2 M betaine [23], 5 - 25 mM tetrapropylammonium chloride [5], 0.5 - 1.5 M 1,2-propanediol, 0.5 - 1.5 M ethyleneglycol [24], 50 - 150 μM 7-deaza-2'-deoxyguanosine 5'-triphosphate [25,26], PCR-enhancing coctail containing 0.3 M D-(+)-trehalose, 0.24 M L-carnitine, and 0.4% Nonidet P-40 (TCN) [27] and antibody-mediated hot start PCR [5] combined with "touchdown" procedure [28,29]. Yet, none of these additives and/or procedures improved PCR to get specific signal determined by agarose gel electrophoresis (data not shown). Interestingly, specific amplicons were observed in PCR mix II supplemented with both 1 M 1,2-propanediol and 0.2 M trehalose (mix II-PT); higher or lower concentrations of these two additives resulted in inhibition of production of the specific amplicons and/or enhanced formation of nonspecific DNA fragments (Figure 2).

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