Theoretical analysis of the kinetics of DNA hybridization with gel-immobilized oligonucleotides

Livshits, M. A.; Mirzabekov, Andrei D.

doi:10.1016/s0006-3495(96)79473-0

Cited by 110 publications

(99 citation statements)

References 20 publications

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“…In this scenario, the effect of free energy penalty associated with mismatched base pairs becomes a fraction of the total free energy of binding (2). This would explain why mismatches associated in shorter sequences promote higher levels of destabilization in a duplex (3,18).…”

Section: Discussionmentioning

confidence: 99%

High-Throughput Detection of Pathogenic Yeasts of the Genus Trichosporon

2004

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The need for a rapid and accurate method for the detection of fungal pathogens has become imperative as the incidence of fungal infections has increased dramatically. Herein, we tested the Luminex 100, a novel flow cytometer, for the detection of the medically important genus Trichosporon. This genus was selected as our proof-of-concept model due to the close phylogenetic relationship between the species. The method, which is based on a nucleotide hybridization assay, consists of a combination of different sets of fluorescent beads covalently bound to species-specific capture probes. Upon hybridization, the beads bearing the target amplicons are classified by their spectral addresses with a 635-nm laser. Quantitation of the hybridized biotinylated amplicon is based on fluorescence detection with a 532-nm laser. We tested in various multiplex formats 48 species-specific and group-specific capture probes designed in the D1/D2 region of ribosomal DNA, internal transcribed spacer regions, and intergenic spacer region. Species-specific biotinylated amplicons were generated with three sets of primers to yield fragments from the three regions. The assay was specific and fast, as it discriminated species differing by 1 nucleotide and required less than 50 min following amplification to process a 96-well plate. The sensitivity of the assay allowed the detection of 10 2 genome molecules in PCRs and 10 7 to 10 8 molecules of biotinylated amplification product. This technology provided a rapid means of detection of Trichosporon species with the flexibility to identify species in a multiplex format by combining different sets of beads.

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Section: Discussionmentioning

confidence: 99%

High-Throughput Detection of Pathogenic Yeasts of the Genus Trichosporon

2004

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“…Solid surface hybridizations with linear DNA and PNA probes have been used to study association and dissociation rates. For example, using immobilized linear DNA oligonucleotide probes, Livshits and Mirzabekov demonstrated that the association rate constant was the same for target and nontarget DNA (27), and Dai et al showed that nontargets exhibited higher dissociation rate constants than targets, which resulted in faster overall kinetics for nontarget (14). In contrast, immobilized linear PNA probes generally exhibited higher association rate constants and lower dissociation rate constants for targets than for nontargets (21).…”

Section: Discussionmentioning

confidence: 99%

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

Morgenroth

Raskin

2008

Appl Environ Microbiol

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The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.Accurate quantification of specific populations in complex microbial communities is important in many areas of microbiology. Today, real-time PCR-based techniques are often used for this purpose (45). Other PCR-based techniques, such as denaturing gradient gel electrophoresis (30) and terminal restriction fragment length polymorphism (29), have also been used to study microbial population dynamics, but they are less suitable as quantitative techniques (52). Oligonucleotide probe-based microbial quantification methods that do not rely on PCR usually target the small subunit rRNA and include quantitative membrane hybridization, quantitative fluorescence in situ hybridization (FISH), and phylogenetic microarrays. While membrane hybridization has been used successfully to study microbial population dynamics in a variety of complex microbial systems (39, 58), this technique is time consuming and labor intensive. Quantitative FISH also has been used effectively to quantify microbial populations (34, 59). However, in some applications, traditional FISH methods do not provide satisfactory quantitative results because of poor cell permeability for oligonucleotide probes (10), low accessibility of target sites in rRNA (18), or poor sensitivity when the cellular rRNA content is low (20). Microarray techniques have the potential to quantify rRNA extracted from environmental samples (16, 23), but reproducibility and specificity issues have not been addressed satisfactorily (37,43).The peptide nucleic acid (PNA) oligomer probe was studied for its hybridization properties for the first time about 15 years ago (15). PNA di...

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“…The theory of hybridization of nucleic acid to gel-immobilized oligonucleotides has been developed [Livshits and Mirzabekov, 1996]. The efficiency of the discrimination depends on the position of the mismatch within the probe, the length of the probe, its AT-content, and reaction conditions.…”

Section: Detection Of Polymorphisms: Hybridizationmentioning

confidence: 99%

“…We took advantage of this fact by developing an algorithm to equalize apparent dissociation temperatures of AT-rich and GCrich duplexes. The former probes are loaded at higher concentration than the latter, facilitating the discrimination on the same chip in similar hybridization conditions [Livshits and Mirzabekov, 1996]. Fotin et al [1998] compared the thermodynamics of duplexes in gel and in solution and found that, although they were qualitatively similar, melting temperatures of the same duplexes in gel were almost 20°C lower than in solution.…”

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confidence: 99%