Improved Efficiency and Robustness in qPCR and Multiplex End-Point PCR by Twisted Intercalating Nucleic Acid Modified Primers

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

A novel FRET pair for detection of parallel DNA triplexes by the LightCycler

<p>Abstract</p> <p>Background</p> <p>Melting temperature of DNA structures can be determined on the LightCycler using quenching of FAM. This method is very suitable for pH independent melting point (Tm) determination performed at basic or neutral pH, as a high throughput alternative to UV absorbance measurements. At acidic pH quenching of FAM is not very suitable, since the fluorescence of FAM is strongly pH dependent and drops with acidic pH.</p> <p>Hoogsteen based parallel triplex helix formation requires protonation of cytosines in the triplex forming strand. Therefore, nucleic acid triplexes show strong pH dependence and are stable only at acidic pH. This led us to establish a new pH independent fluorophore based measuring system on the LightCycler for thermal stability studies of parallel triplexes.</p> <p>Results</p> <p>A novel LightCycler FRET pair labelled with ATTO495 and ATTO647N was established for parallel triplex detection with antiparallel duplex as a control for the general applicability of these fluorophores for Tm determination. The ATTO fluorophores were pH stable from pH 4.5 to 7.5. Melting of triplex and duplex structures were accompanied by a large decrease in fluorescence intensity leading to well defined Tm and high reproducibility. Validation of Tm showed low intra- and inter-assay coefficient of variation; 0.11% and 0.14% for parallel triplex and 0.19% and 0.12% for antiparallel duplex. Measurements of Tm and fluorescence intensity over time and multiple runs showed great time and light stability of the ATTO fluorophores. The variance on Tm determinations was significant lower on the LightCycler platform compared to UV absorbance measurements, which enable discrimination of DNA structures with very similar Tm. Labelling of DNA probes with ATTO fluorophore increased Tm of antiparallel duplexes significantly, but not Tm of parallel triplexes.</p> <p>Conclusions</p> <p>We have established a novel pH independent FRET pair with high fluorescence signals on the LightCycler platform for both antiparallel duplex and parallel triplex formation. The method has been thoroughly validated, and is characterized by an excellent accuracy and reproducibility. This FRET pair is especially suitable for ΔTm and Tm<it/> determinations of pH dependent parallel triplex formation.</p

Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids—TINA

Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy and robust design rules based upon more than 2500 melting points (Tm) determined by FRET. To increase the sensitivity of PT, multiple TINAs should be placed with at least 3 nt in-between or preferable one TINA for each half helixturn and/or whole helixturn. We find that ΔTm of base mismatches on PT is remarkably high (between 7.4 and 15.2°C) compared to antiparallel duplexes (between 3.8 and 9.4°C). The specificity of PT by ΔTm increases when shorter TFOs and higher pH are chosen. To increase ΔTms, base mismatches should be placed in the center of the TFO and when feasible, A, C or T to G base mismatches should be avoided. Base mismatches can be neutralized by intercalation of a TINA on each side of the base mismatch and masked by a TINA intercalating direct 3′ (preferable) or 5′ of it. We predict that TINA stabilized PT will improve the sensitivity and specificity of DNA based clinical diagnostic assays

Increasing the Analytical Sensitivity by Oligonucleotides Modified with Para- and Ortho-Twisted Intercalating Nucleic Acids – TINA

The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5′ and 3′ termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch – in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems

Temporal and Spatial Profiles of Chitinase Expression by Norway Spruce in Response to Bark Colonization by Heterobasidion annosum

Pathogen colonization and transcript levels of three host chitinases, putatively representing classes I, II, and IV, were monitored with real-time PCR after wounding and bark infection by Heterobasidion annosum in 32-year-old trees of Norway spruce (Picea abies) with low (clone 409) or high (clone 589) resistance to this pathogen. Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. At 14 days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for clone 589 but had progressed further into the host tissue in clone 409. Transcript levels of the class II and IV chitinases increased after wounding or inoculation, but the transcript level of the class I chitinase declined after these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in clone 589 than in similar sites in clone 409 3 days after inoculation. This difference was even more pronounced 2 to 6 mm away from the inoculation point, where no infection was yet established, and suggests that the clones differ in the rate of chitinase-related signal perception or transduction. At 14 days after inoculation, these transcript levels were higher in clone 409 than in clone 589, suggesting that the massive upregulation of class II and IV chitinases after the establishment of infection comes too late to reduce or prevent pathogen colonization