Regioselective immobilization of short oligonucleotides to acrylic copolymer gels

Timofeev, Edward N.; Kochetkova, S. V.; Mirzabekov, Andrei D.; Florentiev, V. L.

doi:10.1093/nar/24.16.3142

Cited by 102 publications

(89 citation statements)

References 10 publications

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“…A micromatrix containing 100-by 100-by 20 μm polyacrylamide gel pads fixed on a glass slide and spaced 100 μm from each other was manufactured by photopolymerization [88], and activated as described earlier [89]. Six nl of individual 1mM amino-oligonucleotide solutions was applied to each gel pad containing aldehyde groups [88,90] according to the procedure described earlier [89].…”

Section: Oligonucleotide Microarray Design and Constructionmentioning

confidence: 99%

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Bavykin

Mikhailovich

Zakharyev

et al. 2008

Chemico-Biological Interactions

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Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5 minutes. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect differences in both the genotypes and phenotypes of the B. cereus group organisms. NIH Public Access

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Section: Oligonucleotide Microarray Design and Constructionmentioning

confidence: 99%

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Bavykin

Mikhailovich

Zakharyev

et al. 2008

Chemico-Biological Interactions

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“…Rather than being directly attached to glass, the probes are immobilized in three-dimensional polyacrylamide gel pads affixed to the glass (2,6,9,10,12,13,18,(24)(25)(26)30). The gel pads provide a format suitable for the determination of equilibrium and nonequilibrium dissociation kinetics (i.e., melting profiles) of a large number of probe-target duplexes and for determining the dissociation temperature (T d ), the temperature at which 50% of the duplexes remain during a specified wash period (13,25).…”

mentioning

confidence: 99%

Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays

Urakawa

Fantroussi

Smidt

et al. 2003

Appl Environ Microbiol

138

131

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The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfectmatch probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.DNA microarray technology provides parallel nucleic acid hybridizations for a large number of immobilized oligonucleotides or larger DNA fragments on a small surface area (21). In clinical and environmental microbiology, this technology has been used for assessing gene expression (19), characterizing whole genomes (5), identifying bacteria (8, 10, 28), and monitoring microbial populations (12,22). We anticipate that, in the next several years, the application of DNA microarrays to environmental microbiology will greatly improve the understanding of complex microbial communities, which are typically composed of many microbial species.In general, oligonucleotide DNA microarrays containing 15-to 25-mer oligonucleotide probes provide greater discrimination than microarrays composed of larger PCR-amplified DNA fragments. However, a central challenge to the application of DNA microarrays in environmental microbiology is achieving the specificity needed to resolve complex microbial populations, including discriminating between target and nontarget populations that differ by a single nucleotide (10). This level of specificity is needed to resolve variants of highly conserved genes (e.g., those encoding the rRNAs) and to distinguish between closely related target and nontarget microorganisms.In conventional hybridization assays, single-base-pair discrimination is achieved by adjusting the hybridization conditions (e.g., temperature, ionic strength, or formamide concentration) or washing conditions (dissociation) of the probe-target duplex (31). In DNA microarray assays, however, this approach is difficult to use since one set of hybridization and wash conditions does not provide optimal target discrimination for all probes on the microarray. We therefore have developed an alternative approach that uses differences in thermal dissociation rates of probe-target duplexes to resolve matched and mismatched probe-target duplexes (13,25).The oligonucleotide DNA microarray used ...

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“…After exposing to oxygen plasma for 2 min, the PDMS slab was ready for bonding to the glass plate. The glass substrate with DNA probe arrays was prepared using a photo patterning method [23,24]. The glass plate (75 mm × 75 mm) was firstly treated with bind-silane (Sangon, Shanghai, China) and then aligned to a repel-silane (Sangon, Shanghai, China) treated glass cover of the same size.…”

Section: Design and Fabrication Of CD Microfluidic Chipmentioning

confidence: 99%

Rapid screening of phenylketonuria using a CD microfluidic device

Chen¹,

Zhou²,

Li³

et al. 2011

Journal of Chromatography A

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Regioselective immobilization of short oligonucleotides to acrylic copolymer gels

Cited by 102 publications

References 10 publications

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Discrimination of Bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray

Optimization of Single-Base-Pair Mismatch Discrimination in Oligonucleotide Microarrays

Rapid screening of phenylketonuria using a CD microfluidic device

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