Surpassing Specificity Limits of Nucleic Acid Probes via Cooperativity

Satterfield, Brent C.; Bartosiewicz, Matt; West, Jay; Caplan, Michael R.

doi:10.2353/jmoldx.2010.090056

Cited by 6 publications

(5 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We previously reported on Tentacle Probes (Arcxis Biotechnologies, Pleasanton, CA), which were built based on cooperativity and led to a 100-to 200-fold improvement in kinetics and up to a 10,000fold improvement in concentration-independent specificity. 16,17 However, Tentacle Probes are nonextendable and do not solve the problem of primer-dimers. Note that herein we used cooperativity to make another multiple order of magnitude improvement in PCR, only this time with respect to resistance to amplification of primer-dimers.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Cooperative Primers

Satterfield¹

2014

The Journal of Molecular Diagnostics

Self Cite

View full text Add to dashboard Cite

The increasing need to multiplex nucleic acid reactions presses test designers to the limits of amplification specificity in PCR. Although more than a dozen hot starts have been developed for PCR to reduce primer-dimer formation, none can stop the propagation of primer-dimers once formed. Even a small number of primer-dimers can result in false-negatives and/or false-positives. Herein, we demonstrate a new class of primer technology that greatly reduces primer-dimer propagation, showing successful amplification of 60 template copies with no signal dampening in a background of 150,000,000 primer-dimers. In contrast, normal primers, with or without a hot start, experienced signal dampening with as few as 60 primer-dimers and false-negatives with only 600 primer-dimers. This represents more than a 2.5 million-fold improvement in reduction of nonspecific amplification. We also show how a probe can be incorporated into the cooperative primer, with 2.5 times more signal than conventional fluorescent probes.

show abstract

Section: Discussionmentioning

confidence: 99%

“…Adapting math previously derived for cooperative probes 16,17 and neglecting entropic/enthalpic penalties due to restricting mobility from the linker, the effective equilibrium constant for cooperative primers can be expressed as follows:…”

Section: Oligonucleotide Design and Synthesismentioning

confidence: 99%

Cooperative Primers

Satterfield¹

2014

The Journal of Molecular Diagnostics

Self Cite

View full text Add to dashboard Cite

show abstract

“…As a result, when increasing the number of assistant probes from 1 to 3, the fluorescence intensity increased as the number of assistant probes increased by cooperativity of assistant probes. [34,35] In addition, when the number of assistant probes was increased from 4 to 8, the fluorescence intensity inside E. coli did not change much. From this result, it cannot be expected that detection sensitivity was improved simply by increasing the number of assistant probes added concurrently and that there is a design guideline for designing the assistant probe.…”

Section: Resultsmentioning

confidence: 97%

Fluorescence In Situ Hybridization of 16S rRNA in Escherichia coli Using Multiple Photo‐Cross‐Linkable Probes

Fujimoto

Watanabe

2020

ChemistrySelect

View full text Add to dashboard Cite

RNA forms various secondary structures depending on its sequence in cells; the influence of secondary structures must be considered when detecting, regulating, or manipulating RNA. In this study, we utilized the cooperativity of multiple photo‐cross‐linkable assistant probes to evaluate photo‐cross‐linking for RNAs with complex secondary structures. We succeeded in increasing the fluorescence intensity 39.9 times in the region where detection was difficult owing to the formation of a complex secondary structure in the conventional fluorescence in situ hybridization method. This method enables the detection, regulation, and manipulation of RNA that forms various structures depending on the sequence.

show abstract

“…To enable a separation-free signal transduction that reports on TP exclusively with a minimized response from P, T, and TPP, we design one of the probes as a "tentacle" [43,44]: it includes a hairpin next to the target-binding region. The tentacle probe is labeled with a donor/quencher pair such that when the overlapping fragment is not hybridized to the target (in P or TPP), the hairpin stem is hybridized; and donor and quencher are in close proximity reducing donor's emission (Fig.…”

Section: Probe Engineering Considerations and Different Design Evaluationsmentioning

confidence: 99%

Stoichiometric approach to quantitative analysis of biomolecules: the case of nucleic acids

2021

View full text Add to dashboard Cite

Majority of protocols for quantitative analysis of biomarkers (including nucleic acids) require calibrations and target standards. In this work, we developed a principle for quantitative analysis that eliminates the need for a standard of a target molecule. The approach is based on stoichiometric reporting. While stoichiometry is a simple and robust analytical platform, its utility toward the analysis of biomolecules is very limited due to the lack of general methodologies for detecting the equivalence point. In this work, we engineer a new target/probe-binding model that enables detecting the equivalence point while maintaining an appropriate level of specificity. We establish the probe design principles through theoretical simulations and experimental confirmation. Further, we demonstrate the utility of the stoichiometric analysis via a proof-of-concept system based on oligonucleotide hybridization. Overall, the approach that requires neither standard nor calibration yields quantitative results with an adequate accuracy (> 90-110%) and a high specificity. The principles established in our work are very general and can extend beyond oligonucleotide targets toward quantitative analysis of many other biomolecules such as antibodies and proteins.

show abstract

Surpassing Specificity Limits of Nucleic Acid Probes via Cooperativity

Cited by 6 publications

References 25 publications

Cooperative Primers

Cooperative Primers

Fluorescence In Situ Hybridization of 16S rRNA in Escherichia coli Using Multiple Photo‐Cross‐Linkable Probes

Stoichiometric approach to quantitative analysis of biomolecules: the case of nucleic acids

Contact Info

Product

Resources

About