Fabrication of Microarray of Gel-Immobilized Compounds on a Chip by Copolymerization

Vasiliskov, A.V.; Timofeev, Edward N.; Surzhikov, Sergey A.; Drobyshev, Alexei L.; Shick, V.V.; Mirzabekov, Andrei D.

doi:10.2144/99273rr06

Cited by 78 publications

(54 citation statements)

References 16 publications

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“…Probes having two to five mismatches were also incorporated on the microarray. All probes were synthesized with an amino linker at the 3Ј terminus as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

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confidence: 99%

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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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“…Probes having two to five mismatches were also incorporated on the microarray. All probes were synthesized with an amino linker at the 3Ј terminus as described previously (26).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Urakawa

Fantroussi

Smidt

et al. 2003

Appl Environ Microbiol

138

131

View full text Add to dashboard Cite

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“…Three-dimensional "hydrogel" or gel matrix surfaces (32)(33)(34)(35) may have substantial advantages, but 100-fold improvements over planar surfaces may not be realistic. Multiplexed assays in microflow cells (36 ) or bead-based assays (37 ) may have advantages in terms of efficiency of mixing, but to date, to the best of our knowledge, suspension arrays have not reached the detection limits described here.…”

Section: Mass-sensing Conditions For a Microarray Assay (23 )mentioning

confidence: 99%

Evaluating Sandwich Immunoassays in Microarray Format in Terms of the Ambient Analyte Regime

et al. 2004

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Background: Conceptionally, antibody microarrays are simply multiplexed sandwich immunoassays in a miniaturized format. However, from the amounts of capture antibodies used, it is not apparent whether such assays are ambient analyte (Ekins. Clin Chem 1998;44:2015-30) or mass-sensing devices (Silzel et al. Clin Chem 1998;44: 2036 -43). We evaluated multiplexed microarray sandwich assays for 24 mouse serum proteins in these terms within the boundaries of our experimental setup and based on theoretical considerations of the law of mass action. Methods: Capture antibodies for 24 mouse serum proteins were printed on planar microarray substrates. After incubation with mixtures of purified antigens for 1 or 18 h, mixtures of biotinylated detection antibodies were used. High assay sensitivity was achieved by use of resonance-light-scattering particles for signal generation. Titration curves were generated for assay volumes of 20, 40, and 80 L, and detection limits were calculated and compared. The assays were modeled theoretically based on the amounts of capture antibodies and the assay volumes used. Results: As predicted, experimental variations of the assay volume by up to fourfold did not appreciably affect detection. Even for the most sensitive assay, <2% of the analyte molecules present in the sample were captured and generated signal at the detection limit. However, increasing the sample incubation time from 1 to 18 h on average lowered the detection limit threefold. Conclusions: In our experimental setup, all 24 sandwich microarray assays fulfill the criteria of the "ambient

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“…Because the acrylamide matrix restricts the diffusion of amplification products, double-stranded DNA accumulates around the chromosome, forming two overlapping polonies, each amplified from a different region on the same molecule of DNA. A key feature of this protocol is the use of modified primers in the PCR reaction that covalently attach one strand of the amplified DNA to the acrylamide matrix (19,20). This feature allows us to remove the unattached strand from all polonies by heating and washing the slide, leaving single-stranded templates for subsequent SBE reactions (21)(22)(23).…”

Section: Resultsmentioning

confidence: 99%

Digital genotyping and haplotyping with polymerase colonies

Mitra

Butty

Shendure

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

139

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Polymerase colony (polony) technology amplifies multiple individual DNA molecules within a thin acrylamide gel attached to a microscope slide. Each DNA molecule included in the reaction produces an immobilized colony of double-stranded DNA. We genotype these polonies by performing single base extensions with dye-labeled nucleotides, and we demonstrate the accurate quantitation of two allelic variants. We also show that polony technology can determine the phase, or haplotype, of two singlenucleotide polymorphisms (SNPs) by coamplifying distally located targets on a single chromosomal fragment. We correctly determine the genotype and phase of three different pairs of SNPs. In one case, the distance between the two SNPs is 45 kb, the largest distance achieved to date without separating the chromosomes by cloning or somatic cell fusion. The results indicate that polony genotyping and haplotyping may play an important role in understanding the structure of genetic variation.T he study of genetic variation in the human population has the potential to greatly improve human health, both by predicting susceptibility to disease and guiding choice of therapy. The most common genetic variations in the human population are single-nucleotide polymorphisms (SNPs). By studying candidate genes and performing genome-wide linkage disequilibrium studies, scientists are trying to uncover the ''causative SNP,'' the SNP that alters gene function and thereby increases the risk of disease. It has been shown that deriving haplotypes increases the efficiency of linkage disequilibrium studies (1). Surprisingly, recent studies (2, 3) suggest that haplotypes will also be critical for candidate gene studies. These studies found that for some diseases, there is not one single SNP that is responsible for altering gene function, but instead, multiple SNPs interact to alter the function or expression of a protein (4). These alterations occur only when specific combinations of SNPs are present on the same chromosome, so one must determine the haplotype to find a correlation to the observed phenotype. In these cases, the focus has shifted from a causative SNP to a causative haplotype.The most common approach for determining the haplotype, or phase, of a set of SNPs is computational inference from unphased data (3,(5)(6)(7)(8). Although this methodology has greatly increased the power of both linkage and candidate gene studies, a recent study estimates the error rate to be between 19 and 48%, depending on the algorithm used (6). This error rate presents challenges for the use of this method as a research tool and makes it an unlikely candidate for use in the clinical setting. Recent findings suggest a way to improve the accuracy of haplotype inference. Daly et al. (9) and others (10, 11) have shown that SNPs tend to be inherited in larger haplotype blocks than previously thought, and that there are relatively few variants of each block. This observation has sparked a public effort to characterize all common haplotypes in the human population (12). ...

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Fabrication of Microarray of Gel-Immobilized Compounds on a Chip by Copolymerization

Cited by 78 publications

References 16 publications

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

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

Evaluating Sandwich Immunoassays in Microarray Format in Terms of the Ambient Analyte Regime

Digital genotyping and haplotyping with polymerase colonies

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