Detection of Single Base Alterations in Genomic DNA by Solid Phase Polymerase Chain Reaction on Oligonucleotide Microarrays

Huber, Martin; Losert, Doris; Hiller, Reinhard; Harwanegg, Christian; Mueller, Manfred W.; Schmidt, Wolfgang M.

doi:10.1006/abio.2001.5355

Cited by 47 publications

(29 citation statements)

References 22 publications

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“…10 5 to 10 6 genomes per reaction), which has significant (negative) implications for many environmental monitoring applications. Supplementing the reaction mixture with unbound (gene-specific) primers and allowing the PCR to simultaneously proceed in the liquid and solid phases was one approach to increase analytical sensitivity (34)(35)(36)(37). These methods are an analog of nested PCR, where the first stage of the reaction amplifies DNA in solution and the second stage results in attachment of amplified fragments to immobilized PCR primers with subsequent chain extension.…”

Section: Discussionmentioning

confidence: 99%

Profiling In Situ Microbial Community Structure with an Amplification Microarray

Chandler

Knickerbocker

Bryant

et al. 2013

Appl Environ Microbiol

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The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO 3 ؊ ) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO 3 , but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications.

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Section: Discussionmentioning

confidence: 99%

Profiling In Situ Microbial Community Structure with an Amplification Microarray

Chandler

Knickerbocker

Bryant

et al. 2013

Appl Environ Microbiol

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“…This not only avoided the production of nonspecific solid-phase PCR products but it also allowed the use of a single PCR program for both the liquidand the solid-phase PCRs. High annealing temperatures also made it possible to combine the annealing and extension stages of the on-chip PCR (17,18), resulting in short cycle times (approximately 75 min with a PTC 200 In Situ slide thermocycler). On-chip PCRs were conducted with DNA extracts of all 22 bacterial type culture strains used in this study.…”

Section: Resultsmentioning

confidence: 99%

“…This not only avoids laborious sample preparation steps (only a single pipetting step is virtually needed to launch the reaction from a preprepared master mixture), but it also represents an important benefit with respect to the prevention of potential sample contamination. Due to the exquisite specificity of the on-chip amplification reaction (17), single-base alterations in the template DNAs can be detected. This is especially important for the identification of species which show only minor differences within their 23S rDNA sequences, as is the case within the family Enterobacteriaceae (5).…”

Section: Discussionmentioning

confidence: 99%

“…Because the specificity and efficiency of hybridization are difficult to control in a high-density array format, enzyme-based microarray approaches like minisequencing (28), solid-phase primer elongation (29), and solid-phase PCR (1, 43) have been developed. A one-step method for the analysis of genomic sequence variations has been presented previously (17,18), based on a combination of on-chip PCR and simultaneous nested amplification with allele-specific oligonucleotide primers tethered to a glass solid support (glass chip). In this study, we extended this approach by developing a DNA chip for the identification of the common bacteria causing abortion and infertility in mares.…”

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Microarray-Based Identification of Bacteria in Clinical Samples by Solid-Phase PCR Amplification of 23S Ribosomal DNA Sequences

Mitterer

Huber

Leidinger³

et al. 2004

J Clin Microbiol

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The rapid identification of the bacteria in clinical samples is important for patient management and antimicrobial therapy. We describe a DNA microarray-based PCR approach for the quick detection and identification of bacteria from cervical swab specimens from mares. This on-chip PCR method combines the amplification of a variable region of bacterial 23S ribosomal DNA and the simultaneous sequence-specific detection on a solid phase. The solid phase contains bacterial species-specific primers covalently bound to a glass support. During the solid-phase amplification reaction the polymerase elongates perfectly matched primers and incorporates biotin-labeled nucleotides. The reaction products are visualized by streptavidin-cyanine 5 staining, followed by fluorescence scanning. This procedure successfully identified from pure cultures 22 bacteria that are common causes of abortion and sterility in mares. Using the on-chip PCR method, we also tested 21 cervical swab specimens from mares for the presence of pathogenic bacteria and compared the results with those of conventional bacteriological culture methods. Our method correctly identified the bacteria in 12 cervical swab samples, 8 of which contained more than one bacterial species. Due to the higher sensitivity of the on-chip PCR, this method identified bacteria in five cervical swab samples which were not detected by the conventional identification procedure. Our results show that this method will have great potential to be incorporated into the routine microbiology laboratory.

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“…12 An independent cohort of researchers has been working on implementation of SP-PCR to amplification of specific targets on microarray. The suitability of microarray-based SP-PCR for detection of mutations and polymorphisms, [13][14][15][16] detection of toxin genes and microorganisms, 13,[17][18][19][20] eukaryotic expression profiling, 20 methylation analysis, 21 and generation a͒ of extremely long microarray probes 22 has been successfully demonstrated. Some of these works utilized the traditional immobilization of solid phase primers on a functionalized glass surface, while the others used a polyacrylamide gel as a support.…”

Section: Introductionmentioning

confidence: 99%

The role of DNA diffusion in solid phase polymerase chain reaction with gel-immobilized primers in planar and capillary microarray format

Drobyshev

Nasedkina

Zakharova³

2009

Biomicrofluidics

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The solid phase polymerase chain reaction ͑PCR͒ on a gel-based microarray system was studied under various durations of individual stages of the PCR cycle and spatial restriction of the reaction volume. Combining the experimental study with numerical modeling, we demonstrated that the diffusion of the PCR product in and out of a microarray element during the annealing and melting stages, respectively, is the main factor responsible for distinctive features of the studied type of PCR. The restriction of reaction volume leads to faster PCR signal growth. Particularly, the capillary array, whereby gel-based microarray elements are located on a glass bar inserted into capillary chamber, was found to be a suitable format for the development of the platform.

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Detection of Single Base Alterations in Genomic DNA by Solid Phase Polymerase Chain Reaction on Oligonucleotide Microarrays

Cited by 47 publications

References 22 publications

Profiling In Situ Microbial Community Structure with an Amplification Microarray

Profiling In Situ Microbial Community Structure with an Amplification Microarray

Microarray-Based Identification of Bacteria in Clinical Samples by Solid-Phase PCR Amplification of 23S Ribosomal DNA Sequences

The role of DNA diffusion in solid phase polymerase chain reaction with gel-immobilized primers in planar and capillary microarray format

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