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

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 5min. 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 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.

Emerging array-based technologies in proteomics.

Microarray format is the method of choice for high-throughput hybridization of nucleic acids. Lately, microarray strategy has been applied to broad studies of interactions of proteins with other molecules. The diversity of these interactions and variations of the properties of proteins present new challenges to microchip technology. Significant achievements have been reported. In particular, three-dimensional (gel-based) chips can be manufactured with limited resources and offer flexibility, capacity, homogeneous solvent environment and the ability to record processes in real time and in variable conditions.

Clinical screening of gene rearrangements in childhood leukemia by using a multiplex polymerase chain reaction-microarray approach.

PURPOSE: Currently, many forms of leukemia are considered potentially curable, with prognosis and clinical outcome strongly dependent on the underlying molecular pathophysiology. A substantial number of leukemia patients harbor nonrandom karyotypic abnormalities that define subgroups with unique biological and clinical features. For detection of these types of gene rearrangements, a combination of multiplex RT-PCR with hybridization on oligonucleotide gel array was presented previously, which identified five chromosomal translocations with fusion variants. In the present study, additional clinically relevant translocations were included in our analysis using a second generation of microarrays. We also expanded significantly on the clinical correlation of our findings. EXPERIMENTAL DESIGN: An oligonucleotide microarray was designed for hybridization with products of a multiplex RT-PCR to identify the following translocations: t(9;22)p190, t(4;11), t(12;21), t(1;19), typical for acute lymphoblastic leukemia; t(9;22)p210 for chronic myeloid leukemia; and t(8;21), t(15;17), inv16, typical for acute myeloblastic leukemia. RESULTS: To demonstrate the potential clinical application of the method, 247 cases of childhood leukemia were screened, and the above-mentioned gene rearrangements were found in 30% of cases. The sensitivity and specificity of the assay is comparable with the RT-PCR technique, so that it can be used to follow minimal residual disease. The feasibility of an additional refinement of the method, on-chip-multiplex PCR, has been successfully demonstrated by identifying a common translocation, t(9;22), in chronic myeloid leukemia. CONCLUSIONS: Our data suggest that the microarray-based assay can be an effective and reliable tool in the clinical screening of leukemia patients for the presence of specific gene rearrangements with important diagnostic and prognostic implications. The method is amenable for automation and high-throughput analysis.

Analysis of SNPs and other genomic variations using gel-based chips.

Application of microarrays for the analysis of point mutations and SNPs in genomic DNAs is currently under intensive development. Various technologies are being investigated, employing enzymatic, chemical, and physical tools [for review, see Tillib and Mirzabekov, 2001]. Our current approach is based on the use of IMAGE chips (immobilized microarrays of gel elements) consisting of an array of gel pads attached to a hydrophobic glass surface. The gel pads range in size from picoliters to nanoliters and are used for immobilization of oligonucleotide probes, as well as miniature test tubes for chemical or enzymatic reactions with tethered compounds. Nucleic acids are hybridized, fractionated, modified, and subjected to enzymatic reactions inside the pads. All steps of sequence analysis (PCR-amplification, activation or release of primers and products, DNA extension, hybridization, and reading of the results) can be performed within the same pad. A flexible and inexpensive technology platform enables one to monitor processes in the arrays in both real time and steady-state. Identification of SNPs, microsequencing, and other specific tasks are easily performed. In particular, stacking interactions with short oligonucleotides enhance the capability of high-throughput screening. The IMAGE chips can be analyzed using a variety of equipment, from a dedicated multi-color fluorescent microscope or MALDI-spectrometer to an inexpensive portable analyzer suitable for field conditions. Customized gel-based chips were successfully used for screening of SNPs in a broad range of biologically meaningful genes.

Fluorescence data analysis on gel-based biochips.

A series of biochip readers developed for gel-based biochips includes three imaging models and a novel nonimaging biochip scanner. The imaging readers, ranging from a research-grade versatile reader to a simple portable one, use wide-field objectives and 12-bit digital large-coupled device cameras for parallel addressing of multiple array elements. This feature is valuable for monitoring the kinetics of sample interaction with immobilized probes. Depending on the model and the label used, the sensitivity of these readers approaches 0.3 amol of a labeled sample per gel element. In the selective scanner, both the spot size of the excitation laser beam and the detector field of view match the size of the biochip array elements so that the whole row of the array can be read in a single scan. The portable version reads 50-mm long, 150-element, one-dimensional arrays in 5 s. With a dynamic range of 4000:1, a sensitivity of 1-5 amol of a labeled sample per gel element, and a data format facilitating online processing, the scanner is an attractive, inexpensive solution for biomedical diagnostics. Fluorophores for sample labeling were compared experimentally in terms of detection sensitivity, influence on duplex stability, and suitability for multilabel analysis and thermodynamic studies. Texas Red and tetracarboxyphenylporphyn proved to be the best choice for two-wavelength analysis using the imaging readers.

Identification of chromosomal translocations in leukemias by hybridization with oligonucleotide microarrays.

BACKGROUND AND OBJECTIVES: Identification of chromosomal rearrangements is important for a precise risk-stratified diagnosis of hematologic malignancies. As the number of known translocations, specific for different types of leukemia increases, it takes ever more time and increasing amounts of patient's material to screen a single patient with individual polymerase chain reactions (PCR). The aim of this study was to develop a new approach combining specificity with high-throughput sufficient for rapid screening of clinical samples for the presence of numerous translocations. DESIGN AND METHODS: We designed an oligonucleotide microarray and used hybridization with microarrays in combination with multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay for accurate and rapid identification of some major leukemias. The following translocations were used as prototypic: t(9;22) p210 and p190, t(4;11), t(12;21), and t(15;17). This approach was tested on five different cell cultures carrying translocations and on 22 clinical samples from leukemic patients. RESULTS: Distinctive hybridization signals were obtained for all types of chimeric transcripts from cell lines with translocations. Both the type of translocation and the splice variant were determined. The data demonstrated high specificity and reproducibility of the method. Analysis of the 22 clinical samples using the microarray-based approach showed complete agreement with standard PCR analysis. INTERPRETATION AND CONCLUSIONS: Our data suggest that oligonucleotide microarrays can be used as an efficient, alternative approach to the traditional post-PCR Southern blot analysis. The oligonucleotide microarray approach appears suitable for clinical screening of major risk-stratifying translocations in patients with leukemia.

Radical-generating coordination complexes as tools for rapid and effective fragmentation and fluorescent labeling of nucleic acids for microchip hybridization.

DNA microchip technology is a rapid, high-throughput method for nucleic acid hybridization reactions. This technology requires random fragmentation and fluorescent labeling of target nucleic acids prior to hybridization. Radical-generating coordination complexes, such as 1,10-phenanthroline-Cu(II) (OP-Cu) and Fe(II)-EDTA (Fe-EDTA), have been commonly used as sequence nonspecific "chemical nucleases" to introduce single-strand breaks in nucleic acids. Here we describe a new method based on these radical-generating complexes for random fragmentation and labeling of both single- and double-stranded forms of RNA and DNA. Nucleic acids labeled with the OP-Cu and the Fe-EDTA protocols revealed high hybridization specificity in hybridization with DNA microchips containing oligonucleotide probes selected for identification of 16S rRNA sequences of the Bacillus group microorganisms. We also demonstrated cDNA- and cRNA-labeling and fragmentation with this method. Both the OP-Cu and Fe-EDTA fragmentation and labeling procedures are quick and inexpensive compared to other commonly used methods. A column-based version of the described method does not require centrifugation and therefore is promising for the automation of sample preparations in DNA microchip technology as well as in other nucleic acid hybridization studies.

Species-level identification of orthopoxviruses with an oligonucleotide microchip.

A method for species-specific detection of orthopoxviruses pathogenic for humans and animals is described. The method is based on hybridization of a fluorescently labeled amplified DNA specimen with the oligonucleotide DNA probes immobilized on a microchip (MAGIChip). The probes identify species-specific sites within the crmB gene encoding the viral analogue of tumor necrosis factor receptor, one of the most important determinants of pathogenicity in this genus of viruses. The diagnostic procedure takes 6 h and does not require any sophisticated equipment (a portable fluorescence reader can be used).

Radioisotopic, culture-based, and oligonucleotide microchip analyses of thermophilic microbial communities in a continental high-temperature petroleum reservoir.

Activity measurements by radioisotopic methods and cultural and molecular approaches were used in parallel to investigate the microbial biodiversity and its physiological potential in formation waters of the Samotlor high-temperature oil reservoir (Western Siberia, Russia). Sulfate reduction with rates not exceeding 20 nmol of H(2)S liter(-1) day(-1) occurred at 60 and 80 degrees C. In upper horizons (AB, A, and B), methanogenesis (lithotrophic and/or acetoclastic) was detected only in wells in which sulfate reduction did not occur. In some of the wells from deeper (J) horizons, high-temperature sulfate reduction and methanogenesis occurred simultaneously, the rate of lithotrophic methanogenesis exceeding 80 nmol of CH(4) liter(-1) day(-1). Enrichment cultures indicated the presence of diverse physiological groups representing aerobic and anaerobic thermophiles and hyperthermophiles; fermentative organotrophs were predominant. Phylogenetic analyses of 15 isolates identified representatives of the genera Thermotoga, Thermoanaerobacter, Geobacillus, Petrotoga, Thermosipho, and Thermococcus, the latter four being represented by new species. Except for Thermosipho, the isolates were members of genera recovered earlier from similar habitats. DNA obtained from three samples was hybridized with a set of oligonucleotide probes targeting selected microbial groups encompassing key genera of thermophilic bacteria and archaea. Oligonucleotide microchip analyses confirmed the cultural data but also revealed the presence of several groups of microorganisms that escaped cultivation, among them representatives of the Aquificales/Desulfurobacterium-Thermovibrio cluster and of the genera Desulfurococcus and Thermus, up to now unknown in this habitat. The unexpected presence of these organisms suggests that their distribution may be much wider than suspected.