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Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers.

Schneider UV, Mikkelsen ND, Lindqvist A, Okkels LM, Jøhnk N, Lisby G - PLoS ONE (2012)

Bottom Line: In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers.For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets.Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness.

View Article: PubMed Central - PubMed

Affiliation: QuantiBact A/S, Hvidovre, Denmark. uvs@quantibact.com

ABSTRACT
We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.

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Efficiency curves for unmodified and 5′-o-TINA modified primers.All experiments were conducted at an annealing temperature (Ta) of 66.0°C and primer concentrations (Cprimers) of unmodified and o-TINA modified primers were compared on the same plate. The melting curves corresponding to the amplification curves used for efficiency curve determination are included for the lowest Cprimers that allows an efficiency of 100% for unmodified and 5′-o-TINA modified primers.
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pone-0038451-g002: Efficiency curves for unmodified and 5′-o-TINA modified primers.All experiments were conducted at an annealing temperature (Ta) of 66.0°C and primer concentrations (Cprimers) of unmodified and o-TINA modified primers were compared on the same plate. The melting curves corresponding to the amplification curves used for efficiency curve determination are included for the lowest Cprimers that allows an efficiency of 100% for unmodified and 5′-o-TINA modified primers.

Mentions: The unmodified DNA primers were found to allow for a qPCR efficiency of 100%, when a Cprimers of 400 nM or more was used at a Ta of 66°C or less (Figure 2). A similar (100%) qPCR efficiency at 66°C was obtained with a 5′-o-TINA modified Cprimers of 200 nM or more (Figure 2). For both unmodified and 5′-o-TINA modified primers, the qPCR efficiency decreased equally as Cprimers were further incrementally decreased, as expected (Figure 2). To address the effect of different Cprimers on the quantification cycle threshold (Cq) [20], we compared unmodified and 5′-o-TINA modified Cprimers in the range 50 to 800 nM and a Ta from 58 to 72°C with 1000 copies of target per reaction (Figure S2). At all Cprimers and Ta, the Cq of 5′-o-TINA modified primers was similar to or lower compared to unmodified primers. Cq indisputably increased at lower Cprimers and increasing Ta for both unmodified and 5′-o-TINA modified primers. The difference in Cq between the unmodified primers and the 5′-o-TINA modified primers indisputably increased in favor of the 5′ o-TINA modified primers as Ta was increased towards 72°C, e.g. at 68°C, a DNA Cprimers of 800 nM reached the minimal Cq level, whereas a 5′-o-TINA Cprimers of 300 nM or higher reached the same Cq (Figure S2). As the Ta increased from 58°C towards 72°C, we observed that the minimal Cq level was lowered. This was caused by a decrease in Cq threshold level, since the background signals of the assay decreased by increasing Ta and thereby increased the signal-to-noise ratio of the assay (Figure S2). We used temperature gradient experiments with inter-assay normalization to evaluate the effect of Ta on Cq at different Cprimers for both unmodified and 5′-o-TINA modified primers (Figure 3). For unmodified primers at Cprimers of 400 nM or more, the minimal Cq was observed at a Ta below 69°C (Figure 3a). For 5′-o-TINA modified primers, a similar Cq was observed at Cprimers of 200 nM or more and at a Ta below 72°C (Figure 3b). We conclude that 5′-o-TINA modified primers compared to unmodified DNA primers sustain a qPCR efficiency of 100% at significantly lower Cprimers, higher Ta and combinations of both.


Improved efficiency and robustness in qPCR and multiplex end-point PCR by twisted intercalating nucleic acid modified primers.

Schneider UV, Mikkelsen ND, Lindqvist A, Okkels LM, Jøhnk N, Lisby G - PLoS ONE (2012)

Efficiency curves for unmodified and 5′-o-TINA modified primers.All experiments were conducted at an annealing temperature (Ta) of 66.0°C and primer concentrations (Cprimers) of unmodified and o-TINA modified primers were compared on the same plate. The melting curves corresponding to the amplification curves used for efficiency curve determination are included for the lowest Cprimers that allows an efficiency of 100% for unmodified and 5′-o-TINA modified primers.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368873&req=5

pone-0038451-g002: Efficiency curves for unmodified and 5′-o-TINA modified primers.All experiments were conducted at an annealing temperature (Ta) of 66.0°C and primer concentrations (Cprimers) of unmodified and o-TINA modified primers were compared on the same plate. The melting curves corresponding to the amplification curves used for efficiency curve determination are included for the lowest Cprimers that allows an efficiency of 100% for unmodified and 5′-o-TINA modified primers.
Mentions: The unmodified DNA primers were found to allow for a qPCR efficiency of 100%, when a Cprimers of 400 nM or more was used at a Ta of 66°C or less (Figure 2). A similar (100%) qPCR efficiency at 66°C was obtained with a 5′-o-TINA modified Cprimers of 200 nM or more (Figure 2). For both unmodified and 5′-o-TINA modified primers, the qPCR efficiency decreased equally as Cprimers were further incrementally decreased, as expected (Figure 2). To address the effect of different Cprimers on the quantification cycle threshold (Cq) [20], we compared unmodified and 5′-o-TINA modified Cprimers in the range 50 to 800 nM and a Ta from 58 to 72°C with 1000 copies of target per reaction (Figure S2). At all Cprimers and Ta, the Cq of 5′-o-TINA modified primers was similar to or lower compared to unmodified primers. Cq indisputably increased at lower Cprimers and increasing Ta for both unmodified and 5′-o-TINA modified primers. The difference in Cq between the unmodified primers and the 5′-o-TINA modified primers indisputably increased in favor of the 5′ o-TINA modified primers as Ta was increased towards 72°C, e.g. at 68°C, a DNA Cprimers of 800 nM reached the minimal Cq level, whereas a 5′-o-TINA Cprimers of 300 nM or higher reached the same Cq (Figure S2). As the Ta increased from 58°C towards 72°C, we observed that the minimal Cq level was lowered. This was caused by a decrease in Cq threshold level, since the background signals of the assay decreased by increasing Ta and thereby increased the signal-to-noise ratio of the assay (Figure S2). We used temperature gradient experiments with inter-assay normalization to evaluate the effect of Ta on Cq at different Cprimers for both unmodified and 5′-o-TINA modified primers (Figure 3). For unmodified primers at Cprimers of 400 nM or more, the minimal Cq was observed at a Ta below 69°C (Figure 3a). For 5′-o-TINA modified primers, a similar Cq was observed at Cprimers of 200 nM or more and at a Ta below 72°C (Figure 3b). We conclude that 5′-o-TINA modified primers compared to unmodified DNA primers sustain a qPCR efficiency of 100% at significantly lower Cprimers, higher Ta and combinations of both.

Bottom Line: In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers.For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets.Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness.

View Article: PubMed Central - PubMed

Affiliation: QuantiBact A/S, Hvidovre, Denmark. uvs@quantibact.com

ABSTRACT
We introduce quantitative polymerase chain reaction (qPCR) primers and multiplex end-point PCR primers modified by the addition of a single ortho-Twisted Intercalating Nucleic Acid (o-TINA) molecule at the 5'-end. In qPCR, the 5'-o-TINA modified primers allow for a qPCR efficiency of 100% at significantly stressed reaction conditions, increasing the robustness of qPCR assays compared to unmodified primers. In samples spiked with genomic DNA, 5'-o-TINA modified primers improve the robustness by increased sensitivity and specificity compared to unmodified DNA primers. In unspiked samples, replacement of unmodified DNA primers with 5'-o-TINA modified primers permits an increased qPCR stringency. Compared to unmodified DNA primers, this allows for a qPCR efficiency of 100% at lowered primer concentrations and at increased annealing temperatures with unaltered cross-reactivity for primers with single nucleobase mismatches. In a previously published octaplex end-point PCR targeting diarrheagenic Escherichia coli, application of 5'-o-TINA modified primers allows for a further reduction (>45% or approximately one hour) in overall PCR program length, while sustaining the amplification and analytical sensitivity for all targets in crude bacterial lysates. For all crude bacterial lysates, 5'-o-TINA modified primers permit a substantial increase in PCR stringency in terms of lower primer concentrations and higher annealing temperatures for all eight targets. Additionally, crude bacterial lysates spiked with human genomic DNA show lesser formation of non-target amplicons implying increased robustness. Thus, 5'-o-TINA modified primers are advantageous in PCR assays, where one or more primer pairs are required to perform at stressed reaction conditions.

Show MeSH
Related in: MedlinePlus