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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.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.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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Ortho-TINA (o-TINA) modification attached to the 5′-end of the primer sequence.
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pone-0038451-g001: Ortho-TINA (o-TINA) modification attached to the 5′-end of the primer sequence.

Mentions: Twisted Intercalating Nucleic Acid (TINA) is a novel group of nucleic acid intercalating molecules (Figure 1) [4], [5]. We have previously shown that ortho-TINA (o-TINA) modified oligonucleotides can improve the analytical sensitivity of a hybridization capture assay without increasing the cross-reactivity [6]. For optimal thermal stabilization of antiparallel duplex helixes, the o-TINA molecules are added by covalent linkage at the 5′- and/or 3′- terminal ends of oligonucleotide sequences [6]. This is different from artificial nucleic acids and nucleic acid mimics, such as Locked Nucleic Acid (LNA) and Peptide Nucleic Acid (PNA) which are placed internally as nucleotide substitutions in the oligonucleotide sequence, and which are well known to inhibit the DNA polymerase when placed in PCR primers [7], [8]. We have tested 5′-o-TINA modified oligonucleotides as PCR primers, since the observed improved analytical sensitivity in oligonucleotide hybridization could potentially translate into improved efficacy of the PCR reaction.


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)

Ortho-TINA (o-TINA) modification attached to the 5′-end of the primer sequence.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038451-g001: Ortho-TINA (o-TINA) modification attached to the 5′-end of the primer sequence.
Mentions: Twisted Intercalating Nucleic Acid (TINA) is a novel group of nucleic acid intercalating molecules (Figure 1) [4], [5]. We have previously shown that ortho-TINA (o-TINA) modified oligonucleotides can improve the analytical sensitivity of a hybridization capture assay without increasing the cross-reactivity [6]. For optimal thermal stabilization of antiparallel duplex helixes, the o-TINA molecules are added by covalent linkage at the 5′- and/or 3′- terminal ends of oligonucleotide sequences [6]. This is different from artificial nucleic acids and nucleic acid mimics, such as Locked Nucleic Acid (LNA) and Peptide Nucleic Acid (PNA) which are placed internally as nucleotide substitutions in the oligonucleotide sequence, and which are well known to inhibit the DNA polymerase when placed in PCR primers [7], [8]. We have tested 5′-o-TINA modified oligonucleotides as PCR primers, since the observed improved analytical sensitivity in oligonucleotide hybridization could potentially translate into improved efficacy of the PCR reaction.

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.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.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