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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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Comparison of primers in samples spiked with human genomic DNA (gDNA).(a-c) unmodified DNA primers and (d-f) 5′-o-TINA modified primers. Each ○ on the efficiency curves represents one threshold cycle (Cq) determination on an amplification curve with a corresponding melting curve, reported as the first derivative. Cq determinations highlighted in red would normally have been excluded based on the amplification curve and melting curve evaluation. (a, d) Unspiked samples. (b, e) All samples and negative controls spiked with 10 ng gDNA. (c, f) All samples and negative controls spiked with 100 ng gDNA. A uniform primer concentration of 200 nM was used in all samples and negative controls. The annealing temperatures for unmodified and o-TINA primers were 60.0°C and 66.0°C, respectively.
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pone-0038451-g004: Comparison of primers in samples spiked with human genomic DNA (gDNA).(a-c) unmodified DNA primers and (d-f) 5′-o-TINA modified primers. Each ○ on the efficiency curves represents one threshold cycle (Cq) determination on an amplification curve with a corresponding melting curve, reported as the first derivative. Cq determinations highlighted in red would normally have been excluded based on the amplification curve and melting curve evaluation. (a, d) Unspiked samples. (b, e) All samples and negative controls spiked with 10 ng gDNA. (c, f) All samples and negative controls spiked with 100 ng gDNA. A uniform primer concentration of 200 nM was used in all samples and negative controls. The annealing temperatures for unmodified and o-TINA primers were 60.0°C and 66.0°C, respectively.

Mentions: As 5′-o-TINA modified primers allow for higher Ta and lower Cprimers in a qPCR assay without compromising the cross-reactivity of the primers, we would expect that primer cross-reactivity with non-target sequences in clinical samples would diminish compared to unmodified primers. Figure 4 indeed demonstrates that addition of 10 or 100 ng of human genomic DNA to the assay hampered accurate qPCR efficiency determinations for unmodified primers, whereas 5′-o-TINA modified primers were left unaffected. For non-spiked samples, 200 nM of unmodified primers at a Ta of 60.0°C resulted in a qPCR efficiency of 100%, whereas samples spiked with human genomic DNA resulted in loss of target dilution linearity (qPCR efficiencies far above 100% in combination with very low coefficient of determination (R2) for data used for efficiency curve determinations) and loss of melting curve uniformity at especially lower target concentrations for the unmodified primers, which would normally lead to exclusion of the data before efficiency curve determinations (Figure 4). However, we have included these data to show the difference in qPCR efficiency and robustness between unmodified and 5′-o-TINA modified primers in samples spiked with human genomic DNA. In contrast, 200 nM of 5′-o-TINA modified primers at a Ta of 66.0°C spiked with equal amounts of human genomic DNA only lead to a minor parallel shift in Cq of the target dilution series, due to an increased background and thereby threshold of the assay. Furthermore, only minor changes in the melting curve profiles were observed (Figure 4). To verify the results, we replicated the experiment, spiking the samples with genomic DNA purified from Escherichia coli and observed similar results (Figure S3). The non-target cross-reactivity of the unmodified primers could be eliminated by increasing Cprimers to 400 nM and Ta to 66.0°C (Figure S3).


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)

Comparison of primers in samples spiked with human genomic DNA (gDNA).(a-c) unmodified DNA primers and (d-f) 5′-o-TINA modified primers. Each ○ on the efficiency curves represents one threshold cycle (Cq) determination on an amplification curve with a corresponding melting curve, reported as the first derivative. Cq determinations highlighted in red would normally have been excluded based on the amplification curve and melting curve evaluation. (a, d) Unspiked samples. (b, e) All samples and negative controls spiked with 10 ng gDNA. (c, f) All samples and negative controls spiked with 100 ng gDNA. A uniform primer concentration of 200 nM was used in all samples and negative controls. The annealing temperatures for unmodified and o-TINA primers were 60.0°C and 66.0°C, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038451-g004: Comparison of primers in samples spiked with human genomic DNA (gDNA).(a-c) unmodified DNA primers and (d-f) 5′-o-TINA modified primers. Each ○ on the efficiency curves represents one threshold cycle (Cq) determination on an amplification curve with a corresponding melting curve, reported as the first derivative. Cq determinations highlighted in red would normally have been excluded based on the amplification curve and melting curve evaluation. (a, d) Unspiked samples. (b, e) All samples and negative controls spiked with 10 ng gDNA. (c, f) All samples and negative controls spiked with 100 ng gDNA. A uniform primer concentration of 200 nM was used in all samples and negative controls. The annealing temperatures for unmodified and o-TINA primers were 60.0°C and 66.0°C, respectively.
Mentions: As 5′-o-TINA modified primers allow for higher Ta and lower Cprimers in a qPCR assay without compromising the cross-reactivity of the primers, we would expect that primer cross-reactivity with non-target sequences in clinical samples would diminish compared to unmodified primers. Figure 4 indeed demonstrates that addition of 10 or 100 ng of human genomic DNA to the assay hampered accurate qPCR efficiency determinations for unmodified primers, whereas 5′-o-TINA modified primers were left unaffected. For non-spiked samples, 200 nM of unmodified primers at a Ta of 60.0°C resulted in a qPCR efficiency of 100%, whereas samples spiked with human genomic DNA resulted in loss of target dilution linearity (qPCR efficiencies far above 100% in combination with very low coefficient of determination (R2) for data used for efficiency curve determinations) and loss of melting curve uniformity at especially lower target concentrations for the unmodified primers, which would normally lead to exclusion of the data before efficiency curve determinations (Figure 4). However, we have included these data to show the difference in qPCR efficiency and robustness between unmodified and 5′-o-TINA modified primers in samples spiked with human genomic DNA. In contrast, 200 nM of 5′-o-TINA modified primers at a Ta of 66.0°C spiked with equal amounts of human genomic DNA only lead to a minor parallel shift in Cq of the target dilution series, due to an increased background and thereby threshold of the assay. Furthermore, only minor changes in the melting curve profiles were observed (Figure 4). To verify the results, we replicated the experiment, spiking the samples with genomic DNA purified from Escherichia coli and observed similar results (Figure S3). The non-target cross-reactivity of the unmodified primers could be eliminated by increasing Cprimers to 400 nM and Ta to 66.0°C (Figure S3).

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