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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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Amplification of an octaplex end-point PCR by unmodified primers and 5′-o-TINA modified primers.Data are shown for the D2168 strain entailing the rrs (401 bp), elt (322 bp) and estAh (190 bp) genes. (a) PCR program length of approximately 130 minutes, with a 10-fold target dilution series on crude bacterial lysate to 10,000-fold dilution and a negative control (NC) for unmodified primers and 5′-o-TINA modified primers. (b) Set-up identical to (a), but with a PCR program length of approximately 70 minutes. The red box highlights the lack of amplicons for the estAh gene with unmodified primers. (c) Set-up identical to (b), but additionally spiked with one µg of human genomic DNA per well. Red boxes highlight a non-specific amplicon with a size equaling the ipaH product. (d) 10-fold dilution of strain D2168 with different primer concentrations (Cprimers) of 50, 100, 150, 200, 300 and 400 nM for unmodified primers and 5′-o-TINA modified primers. Red lines are placed at the minimum Cprimers for unmodified primers. (e, f) Increasing annealing temperatures (Ta) of 55.0, 56.6, 57.9, 59.4, 60.8, 62.3, 63.7, 65.2, 66.6, 68.1, 69.5 and 71.0°C on crude bacterial lysate for unmodified primers (e) and 5′-o-TINA modified primers (f). Red lines are placed at the maximum Ta for unmodified primers. In (a-c, e, f), a Cprimers of 200 nM was used for each primer, except the estAh primers for which 400 nM of each primer was used. The PCR program with a length of approximately 70 minutes was used in (b-f).
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pone-0038451-g005: Amplification of an octaplex end-point PCR by unmodified primers and 5′-o-TINA modified primers.Data are shown for the D2168 strain entailing the rrs (401 bp), elt (322 bp) and estAh (190 bp) genes. (a) PCR program length of approximately 130 minutes, with a 10-fold target dilution series on crude bacterial lysate to 10,000-fold dilution and a negative control (NC) for unmodified primers and 5′-o-TINA modified primers. (b) Set-up identical to (a), but with a PCR program length of approximately 70 minutes. The red box highlights the lack of amplicons for the estAh gene with unmodified primers. (c) Set-up identical to (b), but additionally spiked with one µg of human genomic DNA per well. Red boxes highlight a non-specific amplicon with a size equaling the ipaH product. (d) 10-fold dilution of strain D2168 with different primer concentrations (Cprimers) of 50, 100, 150, 200, 300 and 400 nM for unmodified primers and 5′-o-TINA modified primers. Red lines are placed at the minimum Cprimers for unmodified primers. (e, f) Increasing annealing temperatures (Ta) of 55.0, 56.6, 57.9, 59.4, 60.8, 62.3, 63.7, 65.2, 66.6, 68.1, 69.5 and 71.0°C on crude bacterial lysate for unmodified primers (e) and 5′-o-TINA modified primers (f). Red lines are placed at the maximum Ta for unmodified primers. In (a-c, e, f), a Cprimers of 200 nM was used for each primer, except the estAh primers for which 400 nM of each primer was used. The PCR program with a length of approximately 70 minutes was used in (b-f).

Mentions: As seen in Figure 5a, the unmodified primers and 5′-o-TINA modified primers amplified the three specific targets in the D2168 strain with equal analytical sensitivity using the published PCR program from Brandal LT et al. [18]. For both the unmodified and 5′-o-TINA modified primers an amplification product for the rrs internal control gene was amplified in the negative control and the unmodified PCR primers contrary to the 5′-o-TINA modified primers also amplified a non-specific product with a mass below the estAh amplification product in the two highest target concentrations. As the PCR program length was shortened by 60 minutes from a total PCR program length of approximately 130 minutes to approximately 70 minutes, all three specific amplicons could still be observed with similar analytical sensitivity for the 5′-o-TINA modified primers, whereas an estAh amplification product could not be detected by the unmodified primers (red box in Figure 5b). Using the faster PCR program, amplification of the rrs internal control gene in the negative controls was still observed. To ensure that both PCR programs worked equally well for the remaining five targets, we tested the remaining seven bacterial strains. The D2259 strain, which entail the estAh target confirmed the observations for the D2168 strain, whereas the remaining strains showed no difference in amplification, analytical sensitivity or non-specific amplification between unmodified and 5′-o-TINA modified primers connected to the length of the PCR program (Figure S5). For all strains and both unmodified primers and 5′-o-TINA modified primers, the rrs internal control gene was amplified in the negative control. We therefore changed the PCR buffer to our in-house Euro-Optima buffer system and for all eight bacterial strains the negative controls became negative. Unfortunately the Euro-Optima buffer system gave rise to a non-specific product for the unmodified primers with a mass equaling the mass of the stx2 amplification product in strain 55989, D2259, D2260 and D3522 (Figure S5). In the remaining experiments, we continued to use the Qiagen Multiplex PCR Master Mix containing traces of genomic E. coli DNA, but not the virulence factors.


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)

Amplification of an octaplex end-point PCR by unmodified primers and 5′-o-TINA modified primers.Data are shown for the D2168 strain entailing the rrs (401 bp), elt (322 bp) and estAh (190 bp) genes. (a) PCR program length of approximately 130 minutes, with a 10-fold target dilution series on crude bacterial lysate to 10,000-fold dilution and a negative control (NC) for unmodified primers and 5′-o-TINA modified primers. (b) Set-up identical to (a), but with a PCR program length of approximately 70 minutes. The red box highlights the lack of amplicons for the estAh gene with unmodified primers. (c) Set-up identical to (b), but additionally spiked with one µg of human genomic DNA per well. Red boxes highlight a non-specific amplicon with a size equaling the ipaH product. (d) 10-fold dilution of strain D2168 with different primer concentrations (Cprimers) of 50, 100, 150, 200, 300 and 400 nM for unmodified primers and 5′-o-TINA modified primers. Red lines are placed at the minimum Cprimers for unmodified primers. (e, f) Increasing annealing temperatures (Ta) of 55.0, 56.6, 57.9, 59.4, 60.8, 62.3, 63.7, 65.2, 66.6, 68.1, 69.5 and 71.0°C on crude bacterial lysate for unmodified primers (e) and 5′-o-TINA modified primers (f). Red lines are placed at the maximum Ta for unmodified primers. In (a-c, e, f), a Cprimers of 200 nM was used for each primer, except the estAh primers for which 400 nM of each primer was used. The PCR program with a length of approximately 70 minutes was used in (b-f).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368873&req=5

pone-0038451-g005: Amplification of an octaplex end-point PCR by unmodified primers and 5′-o-TINA modified primers.Data are shown for the D2168 strain entailing the rrs (401 bp), elt (322 bp) and estAh (190 bp) genes. (a) PCR program length of approximately 130 minutes, with a 10-fold target dilution series on crude bacterial lysate to 10,000-fold dilution and a negative control (NC) for unmodified primers and 5′-o-TINA modified primers. (b) Set-up identical to (a), but with a PCR program length of approximately 70 minutes. The red box highlights the lack of amplicons for the estAh gene with unmodified primers. (c) Set-up identical to (b), but additionally spiked with one µg of human genomic DNA per well. Red boxes highlight a non-specific amplicon with a size equaling the ipaH product. (d) 10-fold dilution of strain D2168 with different primer concentrations (Cprimers) of 50, 100, 150, 200, 300 and 400 nM for unmodified primers and 5′-o-TINA modified primers. Red lines are placed at the minimum Cprimers for unmodified primers. (e, f) Increasing annealing temperatures (Ta) of 55.0, 56.6, 57.9, 59.4, 60.8, 62.3, 63.7, 65.2, 66.6, 68.1, 69.5 and 71.0°C on crude bacterial lysate for unmodified primers (e) and 5′-o-TINA modified primers (f). Red lines are placed at the maximum Ta for unmodified primers. In (a-c, e, f), a Cprimers of 200 nM was used for each primer, except the estAh primers for which 400 nM of each primer was used. The PCR program with a length of approximately 70 minutes was used in (b-f).
Mentions: As seen in Figure 5a, the unmodified primers and 5′-o-TINA modified primers amplified the three specific targets in the D2168 strain with equal analytical sensitivity using the published PCR program from Brandal LT et al. [18]. For both the unmodified and 5′-o-TINA modified primers an amplification product for the rrs internal control gene was amplified in the negative control and the unmodified PCR primers contrary to the 5′-o-TINA modified primers also amplified a non-specific product with a mass below the estAh amplification product in the two highest target concentrations. As the PCR program length was shortened by 60 minutes from a total PCR program length of approximately 130 minutes to approximately 70 minutes, all three specific amplicons could still be observed with similar analytical sensitivity for the 5′-o-TINA modified primers, whereas an estAh amplification product could not be detected by the unmodified primers (red box in Figure 5b). Using the faster PCR program, amplification of the rrs internal control gene in the negative controls was still observed. To ensure that both PCR programs worked equally well for the remaining five targets, we tested the remaining seven bacterial strains. The D2259 strain, which entail the estAh target confirmed the observations for the D2168 strain, whereas the remaining strains showed no difference in amplification, analytical sensitivity or non-specific amplification between unmodified and 5′-o-TINA modified primers connected to the length of the PCR program (Figure S5). For all strains and both unmodified primers and 5′-o-TINA modified primers, the rrs internal control gene was amplified in the negative control. We therefore changed the PCR buffer to our in-house Euro-Optima buffer system and for all eight bacterial strains the negative controls became negative. Unfortunately the Euro-Optima buffer system gave rise to a non-specific product for the unmodified primers with a mass equaling the mass of the stx2 amplification product in strain 55989, D2259, D2260 and D3522 (Figure S5). In the remaining experiments, we continued to use the Qiagen Multiplex PCR Master Mix containing traces of genomic E. coli DNA, but not the virulence factors.

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