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PubMed2003

Molecular detection and identification of influenza viruses by oligonucleotide microarray hybridization.

Microarrays of virus-specific oligonucleotides may provide a method of screening samples for the presence or absence of a large variety of viruses simultaneously. Influenza viruses are ideal for evaluating such microarrays because of their genetic and host diversity, and the availability of an extensive sequence database. A collection of 476 influenza virus-specific oligonucleotides was spotted onto glass slides as probes. Viral RNAs were reverse transcribed and amplified by PCR, and the products were labeled with cyanine dyes. The presence of viruses and their identities were determined by hybridization. The fluorescence intensities of oligonucleotide spots were highly reproducible within each slide and satisfactorily proportional between experiments. However, the intensities of probe spots completely complementary to target sequences varied from background to saturation. The variations did not correlate with base composition, nucleotide sequence, or internal secondary structures. Therefore, thresholds for determining whether hybridization to a spot should be judged as positive were assigned individually. Considering only positive spots from probes predicted to be monospecific for influenza virus species, subtype, host source, or gene segment, this method made correct identifications at the species, hemagglutinin subtype, and gene segment levels. Monospecific neuraminidase (NA) subtype probes were insufficiently diverse to allow confident NA subtype assignment. Incorporating positive spots from polyspecific probes into the identification scheme gave similar results. Overall, the results demonstrate the potential of microarray-based oligonucleotide hybridization for multiple virus detection.

Animals↗
PubMed2004

Multiplex detection of mutations in clinical isolates of rifampin-resistant Mycobacterium tuberculosis by short oligonucleotide ligation assay on DNA chips.

A new approach, short-oligonucleotide-ligation assay on DNA chip (SOLAC), is developed to detect mutations in rifampin-resistant Mycobacterium tuberculosis. The method needs only four common probes to detect 15 mutational variants of the rpoB gene within 12 h. Fifty-five rifampin-resistant M. tuberculosis isolates were analyzed, resulting in 87.3% accuracy and 83.6% concordance relative to DNA sequencing.

Antibiotics, Antitubercular↗
PubMed2004

Mapping of genomic segments of influenza B virus strains by an oligonucleotide microarray method.

Similar to other segmented RNA viruses, influenza viruses can exchange genome segments and form a wide variety of reassortant strains upon coreplication within a host cell. Therefore, the mapping of genome segments of influenza viruses is essential for understanding their phenotypes. In this work, we have developed an oligonucleotide microarray hybridization method for simultaneous genotyping of all genomic segments of two highly homologous strains of influenza B virus. A few strain-specific oligonucleotide probes matching each of the eight segments of the viral genomes of the B/Beijing/184/93 and B/Shangdong/7/97 strains were hybridized with PCR-amplified fluorescently labeled single-stranded DNA. Even though there were a few mismatches among the genomes of the studied virus strains, microarray hybridization showed highly significant and reproducible discrimination ability and allowed us to determine the origins of individual genomic segments in a series of reassortant strains prepared as vaccine candidates. Additionally, we were able to detect the presence of at least 5% of mixed genotypes in virus stocks even when conventional sequencing methods failed, for example, for the NS segment. Thus, the proposed microarray method can be used for (i) rapid and reliable genome mapping of highly homologous influenza B viruses and (ii) extensive monitoring of influenza B virus reassortants and the mixed genotypes. The array can be expanded by adding new oligoprobes and using more quantitative assays to determine the origin of individual genomic segments in series of reassortant strains prepared as vaccine candidates or in mixed virus populations.

Animals↗
PubMed2005

Use of microelectronic array technology for rapid identification of clinically relevant mycobacteria.

We developed a new method based on the Nanochip microelectronic array technology for identification of various clinically relevant mycobacterial species. PCR-amplified rRNA genes obtained from 270 positive Mycobacteria Growth Indicator Tube cultures were successfully tested by hybridizing them with species-selective probes, and the results agreed with those of conventional identification methods. The system is rapid and accurate and opens new perspectives in clinical diagnostics.

Bacterial Typing Techniques↗
PubMed2005

Efficient discrimination within a Corynebacterium diphtheriae epidemic clonal group by a novel macroarray-based method.

A large diphtheria epidemic in the 1990s in Russia and neighboring countries was caused by a clonal group of closely related Corynebacterium diphtheriae strains (ribotypes Sankt-Peterburg and Rossija). In the recently published complete genome sequence of C. diphtheriae strain NCTC13129, representative of the epidemic clone (A. M. Cerdeno-Tarraga et al., Nucleic Acids Res. 31:6516-6523, 2003), we identified in silico two direct repeat (DR) loci 39 kb downstream and 180 kb upstream of the oriC region, consisting of minisatellite (27- to 36-bp) alternating DRs and variable spacers. We designated these loci DRA and DRB, respectively. A reverse-hybridization macroarray-based method has been developed to study polymorphism (the presence or absence of 21 different spacers) in the larger DRB locus. We name it spoligotyping (spacer oligonucleotide typing), analogously to a similar method of Mycobacterium tuberculosis genotyping. The method was evaluated with 154 clinical strains of the C. diphtheriae epidemic clone from the St. Petersburg area in Russia from 1997 to 2002. By comparison with the international ribotype database (Institut Pasteur, Paris, France), these strains were previously identified as belonging to ribotypes Sankt-Peterburg (n = 79) and Rossija (n = 75). The 154 strains were subdivided into 34 spoligotypes: 14 unique strains and 20 types shared by 2 to 46 strains; the Hunter Gaston discriminatory index (HGDI) was 0.85. DRB locus-based spoligotyping allows fast and efficient discrimination within the C. diphtheriae epidemic clonal group and is applicable to both epidemiological investigations and phylogenetic reconstruction. The results are easy to interpret and can be presented and stored in a user-friendly digital database (Excel file), allowing rapid type determination of new strains.

Bacterial Typing Techniques↗
PubMed2005

Identification of medically important molds by an oligonucleotide array.

Infections caused by fungi have increased in recent years. Accurate and rapid identification of fungal pathogens is important for appropriate treatment with antifungal agents. On the basis of the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 64 species (32 genera) of clinically important filamentous (or dimorphic) fungi. These 64 species included fungi causing superficial, cutaneous, subcutaneous, and invasive infections. The method consisted of PCR amplification of the ITS regions using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species- or group-specific oligonucleotides immobilized on a nylon membrane. Of 397 fungal strains (290 target and 107 nontarget strains) tested, the sensitivity and specificity of the array was 98.3% (285/290) and 98.1% (105/107), respectively. Misidentified strains were usually those belonging to the same genus of the target species or having partial homology with oligonucleotide probes on the membrane. The whole procedure can be finished within 24 h starting from isolated colonies; reproductive structures, which are essential for the conventional identification methods, are not needed. In conclusion, the present array is a powerful tool for identification of clinically important filamentous fungi and may have the potential to be continually extended by adding further oligonucleotides to the array without significantly increasing the cost or complexity.

Fungi↗
PubMed2005

Simultaneous detection of four human pathogenic microsporidian species from clinical samples by oligonucleotide microarray.

Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microarray assay for the simultaneous detection and identification to the species level of four major microsporidian species: Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon hellem, and Encephalitozoon intestinalis. The 18S small-subunit rRNA gene was chosen as the amplification target, labeled with fluorescence dye, and hybridized to a series of species-specific oligonucleotide probes immobilized on a microchip. The specificity and sensitivity of the microarray were clearly demonstrated by the unique hybridization profiles exhibited by each species of microsporidian tested and its ability to detect as few as 10 spores. In order to assess the applicability of this microarray in a clinical setting, we conducted microarray assays of 20 fecal samples from AIDS patients. Twelve of these samples were positive for the presence of microsporidia and could be confidently identified; 11 of them were positive for more than one species. Our results suggested that this microarray-based approach represents an attractive diagnostic tool for high-throughput detection and identification of microsporidian species in clinical and epidemiological investigations.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Human immunodeficiency virus type 1-induced macrophage gene expression includes the p21 gene, a target for viral regulation.

In contrast to CD4+ T cells, human immunodeficiency virus type 1 (HIV-1)-infected macrophages typically resist cell death, support viral replication, and consequently, may facilitate HIV-1 transmission. To elucidate how the virus commandeers the macrophage's intracellular machinery for its benefit, we analyzed HIV-1-infected human macrophages for virus-induced gene transcription by using multiple parameters, including cDNA expression arrays. HIV-1 infection induced the transcriptional regulation of genes associated with host defense, signal transduction, apoptosis, and the cell cycle, among which the cyclin-dependent kinase inhibitor 1A (CDKN1A/p21) gene was the most prominent. p21 mRNA and protein expression followed a bimodal pattern which was initially evident during the early stages of infection, and maximum levels occurred concomitant with active HIV-1 replication. Mechanistically, viral protein R (Vpr) independently regulates p21 expression, consistent with the reduced viral replication and lack of p21 upregulation by a Vpr-negative virus. Moreover, the treatment of macrophages with p21 antisense oligonucleotides or small interfering RNAs reduced HIV-1 infection. In addition, the synthetic triterpenoid and peroxisome proliferator-activated receptor gamma ligand, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO), which is known to influence p21 expression, suppressed viral replication. These data implicate p21 as a pivotal macrophage facilitator of the viral life cycle. Moreover, regulators of p21, such as CDDO, may provide an interventional approach to modulate HIV-1 replication.

Anti-HIV Agents↗
PubMed2006

Exploring the DNA binding interactions of the Kaposi's sarcoma-associated herpesvirus lytic switch protein by selective amplification of bound sequences in vitro.

The lytic switch protein RTA of Kaposi's sarcoma-associated herpesvirus (KSHV) can be targeted to DNA by either direct sequence-specific recognition or via protein-protein interactions with host transcription factors. We have searched for sequences capable of direct RTA binding by screening synthetic oligonucleotide pools and KSHV genomic libraries for RTA-interacting elements, using repeated cycles of in vitro binding followed by amplification of the bound sequences. Multiple low-affinity sequences were recovered from the random pools, with generation of only a weak consensus sequence. The genomic library, by contrast, yielded many biologically relevant fragments, most of which could be shown to interact with RTA in vitro and some of which likely play important regulatory roles in vivo. Surprisingly, the most highly selected fragment came from the promoter of a late gene (gB) and contained at least two direct RTA binding sites, as well as one RBP-Jkappa binding site. This raises the possibility that some late KSHV genes may also be subject to direct RTA regulation, though indirect models are not excluded.

Base Sequence↗
PubMedDate not supplied

[Functionalized nanocomposite coating of a glass surface for oligonucleotide immobilization].

A new type of coating for manufacturing DNA chips was constructed of the basis of an organic-inorganic nanocomposite based on the polyvinylbutyral-tetraethoxysilane copolymer. The organosilicon composite was functionalized by introduction of ethanolamine vinyl ether copolymers, which contain amino groups and anchor vinyloxide units capable of reacting with silanol groups of the nanocomposite. The resulting coatings form a film on glass slides with a high surface density of amino groups (up to 700 groups/nm2) suitable for three-dimensional immobilization of oligonucleotides. The use of bifunctional reagents (e.g., phenylene diisothiocyanate) for the attachment of oligonucleotides bearing amino linkers to the amino-containing surface provides an immobilization density of 0.5-1.6 pmol/mm2. Immobilization with a higher density (10-12 pmol/mm2) was achieved for attachment to amino-containing glass slides upon the use of oligonucleotides containing selectively activated terminal phosphate groups. The activation of oligonucleotides was carried out with the triphenylphosphine-dithiodipyridine pair in the presence of dimethylaminopyridine N-oxide. The resulting DNA chips were shown to be useful in principle for DNA detection.

DNA↗
PubMed2003

Fast large scale oligonucleotide selection using the longest common factor approach.

We present a fast method that selects oligonucleotide probes (such as DNA 25-mers) for microarray experiments on a truly large scale. For example, reliable oligos for human genes can be found within four days, a speedup of one to two orders of magnitude compared to previous approaches. This speed is attained by using the longest common substring as a specificity measure for candidate oligos. We present a space- and time-efficient algorithm, based on a suffix array with additional information, to compute matching statistics (lengths of longest matches) between all candidate oligos and all remaining sequences. With the matching statistics available, we show how to incorporate constraints such as oligo length, melting temperature, and self-complementarity into the selection process at a postprocessing stage. As a result, we can now design custom oligos for any sequenced genome, just as the technology for on-site chip synthesis is becoming increasingly mature.

Algorithms↗
PubMed2004

Computing highly specific and noise-tolerant oligomers efficiently.

The sequencing of the genomes of a variety of species and the growing databases containing expressed sequence tags (ESTs) and complementary DNAs (cDNAs) facilitate the design of highly specific oligomers for use as genomic markers, PCR primers, or DNA oligo microarrays. The first step in evaluating the specificity of short oligomers of about 20 units in length is to determine the frequencies at which the oligomers occur. However, for oligomers longer than about fifty units this is not efficient, as they usually have a frequency of only 1. A more suitable procedure is to consider the mismatch tolerance of an oligomer, that is, the minimum number of mismatches that allows a given oligomer to match a substring other than the target sequence anywhere in the genome or the EST database. However, calculating the exact value of mismatch tolerance is computationally costly and impractical. Therefore, we studied the problem of checking whether an oligomer meets the constraint that its mismatch tolerance is no less than a given threshold. Here, we present an efficient dynamic programming algorithm solution that utilizes suffix and height arrays. We demonstrated the effectiveness of this algorithm by efficiently computing a dense list of numerous oligo-markers applicable to the human genome. Experimental results show that the algorithm runs faster than well-known Abrahamson's algorithm by orders of magnitude and is able to enumerate 65% approximately 76% of qualified oligomers.

Algorithms↗
PubMed2004

Supersequences of masks for oligo-chips.

On a very small surface, a chip, several thousands of oligonucleotides, can be synthesized using a mask technology. Given a set of oligos, the problems tackled here are: What is the minimum number of masks necessary to synthesize one copy of each oligo? How long will be the series of masks if each oligo is synthesized twice, such that the two copies are realized with two completely different series of masks? We establish that, for 20,000 oligos of 20 bases a single copy can be synthesized with around 67 masks and for two copies with less than the double.

Algorithms↗
PubMed1999

Nucleic acid biotechnology.

Driven by advances in the acquisition of genetic sequence information and the ability to manipulate small quantities of nucleic acid, a number of technologies are emerging that exploit nucleic acids for research, diagnostic, and therapeutic utility. In this review, we cover three technologies based on nucleic acids--DNA microarrays, antisense technology, and gene therapy--that are especially promising and may make a substantial impact in the laboratory and in the clinic during the coming years. For each of these areas, an overview of the current status and applications is provided, followed by a discussion of critical issues and challenges to be faced for further advancement of the technology; an emphasis is placed on quantitative and engineering aspects.

Biomedical Engineering↗
PubMed2005

Gene expression profiling of hypoxia signaling in human hepatocellular carcinoma cells.

Cellular, local, and organismal responses to low O2 availability occur during processes such as anaerobic metabolism and wound healing and pathological conditions such as stroke and cancer. These responses include increases in glycolytic activity, vascularization, breathing, and red blood cell production. These responses are mediated in part by the hypoxia-inducible factors (HIFs), which receive information on O2 levels from a group of iron- and O2-dependent hydroxylases. Hypoxia mimics, such as cobalt chloride, nickel chloride, and deferoxamine, act to simulate hypoxia by altering the iron status of these hydroxylases. To determine whether these mimics are appropriate substitutes for the lower O2 tension evoked naturally, we compared transcriptional responses of a Hep3B cell line using high-density oligonucleotide arrays. A battery of core genes was identified that was shared by all four treatments (hypoxia, cobalt, nickel, and deferoxamine) including glycolytic enzymes, cell cycle regulators, and apoptotic genes. Importantly, cobalt, nickel, and deferoxamine influenced transcription of distinct sets of genes that were not affected by cellular hypoxia. These global responses to hypoxia indicate a balancing act between adaptation and programmed cell death and suggest caution in the use of hypoxia mimics as substitutes for the low O2 tension that occurs in vivo.

Algorithms↗
PubMed2004

A comparison of oligonucleotide and cDNA-based microarray systems.

Large-scale public data mining will become more common as public release of microarray data sets becomes a corequisite for publication. Therefore, there is an urgent need to clarify whether data from different microarray platforms are comparable. To assess the compatibility of microarray data, results were compared from the two main types of high-throughput microarray expression technologies, namely, an oligonucleotide-based and a cDNA-based platform, using RNA obtained from complex tissue (human colonic mucosa) of five individuals. From 715 sequence-verified genes represented on both platforms, 64% of the genes matched in "present" or "absent" calls made by both platforms. Calls were influenced by spurious signals caused by Alu repeats in cDNA clones, clone annotation errors, or matched probes that were designed to different regions of the gene; however, these factors could not completely account for the level of call discordance observed. Expression levels in sequence-verified, platform-overlapping genes were not related, as demonstrated by weakly positive rank order correlation. This study demonstrates that there is only moderate overlap in the results from the two array systems. This fact should be carefully considered when performing large-scale analyses on data originating from different microarray platforms.

Aged↗
PubMed2006

Expression profiling of antisense transcripts on DNA arrays.

The majority of mouse genes are estimated to undergo bidirectional transcription; however, their tissue-specific distribution patterns and physiological significance are largely unknown. This is in part due to the lack of methodology to routinely assess the expression of natural antisense transcripts (NATs) on a large scale. Here we tested whether commercial DNA arrays can be used to monitor antisense transcription in mouse kidney and brain. We took advantage of the reversely annotated oligonucleotides on the U74 mouse genome array from Affymetrix that hybridize to NATs overlapping with the sense transcript in the area of the probe set. In RNA samples from mouse kidney and brain, 11.9% and 10.1%, respectively, of 5,652 potential NATs returned positive and about half of the antisense RNAs were detected in both tissues, which was similar to the fraction of sense transcripts expressed in both tissues. Notably, we found that the majority of NATs are related to the sense transcriptome since corresponding sense transcripts were detected for 92.5% (kidney) and 74.5% (brain) of the detected antisense RNAs. Antisense RNA transcription was confirmed by real-time PCR and included additional RNA samples from heart, thymus, and liver. The randomly selected transcripts showed tissue specific expression patterns and varying sense/antisense ratios. The results indicate that antisense transcriptomes are tissue specific, and although pairing of sense/antisense transcripts are known to result in rapid degradation, our data provide proof of principle that the sensitivity of commercial DNA arrays is sufficient to assess NATs in total RNA of whole organs.

Animals↗
PubMed2004

Target ablation-induced regulation of macrophage recruitment into the olfactory epithelium of Mip-1alpha-/- mice and restoration of function by exogenous MIP-1alpha.

The chemokine macrophage inflammatory protein (MIP)-1alpha recruits macrophages to sites of epithelial remodeling. We showed previously that mRNA and protein levels of MIP-1alpha in the olfactory epithelium (OE) increased significantly at 3 days after bilateral olfactory bulbectomy (OBX). The first aim of this study was to investigate the effect of the absence of MIP-1alpha on macrophage recruitment to the OE 3 days after OBX in Mip-1alpha(-/-) mice compared with C57BL/6 mice and to test whether chemokine function could be restored by MIP-1alpha protein injection into Mip-1alpha(-/-) mice. OBX was performed on C57BL/6 and Mip-1alpha(-/-) mice. The mice received six subcutaneous injections at 12-h intervals of either 10 mug/ml MIP-1alpha protein in carrier or carrier only. Macrophage recruitment was evaluated with antibodies to CD68 for all macrophages and F4/80 for activated macrophages. Compared with C57BL/6 mice, at 3 days post-OBX the numbers of CD68(+) and F4/80(+) macrophages were significantly lower in carrier-injected Mip-1alpha(-/-) mice and were comparable in MIP-1alpha protein-injected Mip-1alpha(-/-) mice. The second aim was to determine the identity of genes regulated at 3 days post-OBX in the OE of carrier-injected Mip-1alpha(-/-) mice compared with carrier-injected C57BL/6 mice. Total RNA from the OE was hybridized to Affymetrix microarrays. A number of chemokine-, cytokine-, and growth factor-related genes were significantly regulated in the Mip-1alpha(-/-) mice and were restored in MIP-1alpha protein-injected Mip-1alpha(-/-) mice. The results illustrated that MIP-1alpha played a key role in recruitment of macrophages to the OE and provided insight into the genomic regulation involved in OE remodeling.

Animals↗