Oligonucleotide Biochip Research Articles

Fluorescent Labeling of Oligonucleotide Probes for Double Indicator Microarray Hybridization Analysis

Herein, we present a synthesis of a molecular construct based on the polysulfonated indocarbocyanine dye and the lysine. The chemical structure of the construct obtained enables fluorescent labeling of oligonucleotide probes designed for the immobilization on the oligonucleotide biochips.

Multiplex 16S rRNA-derived geno-biochip for detection of 16 bacterial pathogens from contaminated foods.

Foodborne diseases caused by various pathogenic bacteria occur worldwide. To prevent foodborne diseases and minimize their impacts, it is important to inspect contaminated foods and specifically detect many types of pathogenic bacteria. Several DNA oligonucleotide biochips based on 16S rRNA have been investigated to detect bacteria; however, a mode of detection that can be used to detect diverse pathogenic strains and to examine the safety of food matrixes is still needed. In the present work, a 16S rRNA gene‐derived geno‐biochip detection system was developed after screening DNA oligonucleotide specific capture probes, and it was validated for multiple detection of 16 pathogenic strains that frequently occur with a signature pattern. rRNAs were also used as detection targets directly obtained from cell lysates without any purification and amplification steps in the bacterial cells separated from 8 food matrixes by simple pretreatments. Thus, the developed 16S rRNA‐derived geno‐biochip can be successfully used for the rapid and multiple detection of the 16 pathogenic bacteria frequently isolated from contaminated foods that are important for food safety.

Highly Effective xMAP Multiplex Assay for the Detection and Identification of Hemorrhagic Fever Agents, Including Ebola Virus

Developed has been the oligonucleotide liquid biochip based on xMAP technology, designed for the laboratory detection of particularly dangerous viral pathogens such as Ebola and Marburg filoviruses, and Machupo , Junin, and Lassa arenaviruses. The suggested approach allows for the detection of up to 100 viral genome equivalents in a sample. The sensitivity and specificity of oligonucleotide biochip is 100 % when the laboratory panels of positive and negative samples are used. These results indicate that the xMAP multiplexing for the detection and identification of tropical hemorrhagic fever agents, including Ebola virus, is not inferior to the conventional method such as real-time RT-PCR and can be applied for evaluation of viral load, and further on can easily be expanded for both the analysis of new viral agents and for the detection of critical mutations in viral genomes.

Synthesis of nucleosides containing a photolabile 2-(2-nitrophenyl)propoxycarbonyl group

At present, DNA biochips are widely used in medicine as diagnostic systems [1, 2]. Oligonucleotide biochips are often obtained with the aid of photolabile protecting groups [3–7]. The latter should be stable under the conditions of oligonucleotide synthesis but should be readily removed by photolysis without involving the protected moiety. A widely used photolabile protecting group is the o-nitrobenzyl group [3, 8–10]. The goal of the present work was to extend the series of nucleosides protected by the photolabile 2-(2-nitrophenyl)propoxycarbonyl group and containing readily removable protecting groups in the aromatic heteroring, which can subsequently be used in the design of oligonucleotide biochips. We have synthesized previously unknown nucleosides 4–7. Compounds 4 and 5 were obtained by acylation of 2′-deoxyguanosine and 2′-deoxyadenosine with phenoxyacetyl chlorides 1 and 2, respectively. Nucleosides 6 and 7 protected by photolabile 2-(2-nitrophenyl)propoxycarbonyl groups were synthesized by treatment of compounds 4 and 5, respectively, with 2-(2-nitrophenyl)propyl chloroformate (3) which was prepared as described in [10]. Compounds 6 and 7 were isolated as mixtures of diastereoisomers. ISSN 1070-4280, Russian Journal of Organic Chemistry, 2015, Vol. 51, No. 1, pp. 141–144. © Pleiades Publishing, Ltd., 2015. Original Russian Text © E.B. Nikolaenkova, I.A. Os’kina, V.A. Savel’ev, A.Ya. Tikhonov, V.A. Ryabinin, A.N. Sinyakov, 2015, published in Zhurnal Organicheskoi Khimii, 2015, Vol. 51, No. 1, pp. 142–144.

Molecular and Genetic Research in Tuberculosis Clinical Practice and Epidemiology.

IntroductionTuberculosis (TB) remains a global public health problem. In order for multi-drug resistant tuberculosis (MDR-TB) to be more effectively managed, there is a need for better tools for diagnosis, treatment, and prevention. The decline of TB incidence and mortality in Kazakhstan during last decade was accompanied with consistent growth of MDR-TB. This study aimed to investigate genotype characteristics of Mycobacterium tuberculosis (MT) isolated from TB patients from different regions of the country and its clinical and epidemiological significance.MethodsOver 500 clinical MT isolates from pulmonary TB patients between 2003–2008 were genotyped using spoligotyping, MIRU-VNTR, IS6110 RFLP, and hybridization on an oligonucleotide biochip “TB–biochip.”ResultsOut of 250 isolates with interpretable results, 31 different spoligopatterns were detected. The Beijing genotype was the most predominant lineage detected (71.6%), characterized by heterogenicity on ETR A, B, C, D, and E markers, and 56.6% of them had an allelic profile 42435. The Beijing genotype and dominating variant strains have a high transmission rate, a high rate of primary MDR (associated with infiltrating lung TB and complications), and a high level resistance to rifampicin and izoniazid due to mutation of rpoB531TTG and katG315ACC. MIRU-VNTR–typing by 15 loci of 33 isolates from 13 family TB foci revealed that strains from supposed sources and contact persons completely coincide in only 5 foci in the genomic structure.ConclusionThere is a heterogeneous pool of genotypes that circulate in Kazakhstan, with the Beijing lineage being the most predominant. It appears that at the present stage of circulation, MT Beijing genotype has an endemic character. However, clonal spreading of epidemiologically and clinically significant MDR strains of this genotype is also a serious threat to the population. To increase TB control efficiency and prevent further transmission, it is necessary to compile a modern countrywide system of microbiological monitoring for the agent by use of a computer bank of spoligotyping and MIRU-VNTR-typing profiles of circulating strains.

Synthesis of mixed-sequence oligonucleotides on mesoporous silicon: chemical strategies and material stability.

Rapid screening tests in medical diagnostic and environmental analysis are often based on oligonucleotide biochips. In this paper, we studied the stability of functionalized mesoporous silicon supports in the solid-phase synthesis of oligonucleotides, exploiting several chemical procedures. A 19-mer mixed sequence has been successfully synthesized on aminosilane-modified porous silicon photonic structures. The process and the materials have been characterized by optical reflectivity, atomic force microscopy and high-performance liquid chromatography.

A Facile Inhibitor Screening of Hepatitis C Virus NS3 Protein Using Nanoparticle-Based RNA

Globally, over hundreds of million people are infected with the hepatitis C virus: the global rate of death as a direct result of the hepatitis C virus has increased remarkably. For this reason, the development of efficient drug treatments for the biological effects of the hepatitis C virus is highly necessary. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide can recognize the hepatitis C virus NS3 protein specifically and sensitively. In this study, we elucidated that this biochip can analyze inhibitors to the hepatitis C virus NS3 protein using a nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, 7,8,4'-trihydroxyisoflavone and 6,7,4'-trihydroxyisoflavone demonstrated a remarkable inhibition activity on the hepatitis C virus NS3 protein. Both 7,8,4'-trihydroxyisoflavone and 6,7,4'-trihydroxyisoflavone attenuated the binding affinity in a concentrated manner as evidenced by QDs conjugated RNA oligonucleotide. At a concentration of 0.01 μg·mL−1, 7,8,4'-trihydroxyisoflavone and 6,7,4'-trihydroxyisoflavone showed more than a 30% inhibition activity of a nanoparticle-based RNA oligonucleotide biochip system.

A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide

Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS), and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV) nucleocapsid (N) protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (−)-catechin gallate and (−)-gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (−)-catechin gallate and (−)-gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 μg mL−1, (−)-catechin gallate and (−)-gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.

Identification of plant-derived genetically modified organisms in food and feed using a hydrogel oligonucleotide microchip

A method of multiplex polymerase chain reaction (PCR) followed by hybridization on a hydrogel oligonucleotide biochip was developed for simultaneous identification of ten different transgenic elements of plant DNA in food and feed products. The biochip contained 22 immobilized oligonucleotide probes that were intended for (1) detection of plant DNA, (2) determination of plant species (soybean, maize, potato, and rice), and (3) identification of transgenic elements, including sequences of 35S CaMV, 35S FMV, rice actin gene promoters, nos, 35S CaMV, ocs, pea rbcS1 gene terminators, and bar, gus, and nptII marker genes. The limit of detection was 0.5% for genetically modified (GM) soybean and maize in the analyzed samples. The tests on food and feed products using the developed approach and real-time PCR showed full agreement in determination of transgenic DNA in the samples. The proposed assay can be used for selection of GM samples by screening food and feed products for subsequent quantitative determination of GM component based on the identified transgene.

(−)-Epigallocatechin gallate inhibits hepatitis C virus (HCV) viral protein NS5B

In this study, we elucidated a small molecule inhibitor on viral protein NS5B identified through a high-throughput screening strategy using optical nanoparticle-based RNA oligonucleotide. We have previously shown that quantum dots (QDs)–RNA oligonucleotide can specifically recognize the HCV viral proteins. We have also demonstrated that conjugated QDs–RNA oligonucleotide can specifically and sensitively interact with designed biochips [1,2]. Among the flavonoids examined, (−)-epigallocatechin gallate (EGCG) demonstrated a remarkable inhibition activity on HCV viral protein, NS5B. (−)-Epigallocatechin gallate, at 0.005μgmL−1 or more, concentration-dependently attenuated the binding affinity on a designed biochip as evidenced by QDs–RNA oligonucleotide. At a concentration of 0.1μgmL−1, (−)-epigallocatechin gallate showed a 50% inhibition activity on QDs–RNA oligonucleotide biochip assay. We screened a small molecule inhibitor on the viral protein, NS5B, identified through a high-throughput screening strategy using on-chip optical nanoparticle-based RNA oligonucleotide on chip. In this designed strategy, the convenient and efficient screening and development of an on-chip viral protein inhibitor using a QDs–RNA oligonucleotide assay is achievable with high sensitivity and simplicity. In addition, this platform is expected to be applicable toward the inhibitor screening of other types of diseases.

A simple oligonucleotide biochip capable of rapidly detecting known mitochondrial DNA mutations in Chinese patients with Leber's hereditary optic neuropathy (LHON).

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease. Clinically, no efficient assay protocols have been available. In this study, we aimed to develop an oligonucleotide biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing LHON and to investigate frequencies of LHON relevant variants in Anhui region of China. Thirty-two pairs of oligonucleotide probes matched with the mutations potentially linked to LHON were covalently immobilized. Cy5-lablled targets were amplified from blood DNA samples by a multiplex PCR method. Two kinds of primary mutations 11778 G > A and 14484 T > C from six confirmed LHON patients were interrogated to validate this biochip format. Further, fourteen Chinese LHON pedigrees and twenty-five unrelated healthy individuals were investigated by the LHON biochip, direct sequencing and pyrosequencing, respectively. The biochip was found to be able efficiently to discriminate homoplasmic and heteroplasmic mtDNA mutations in LHON. Biochip analysis revealed that twelve of eighteen LHON symptomatic cases from the 14 Chinese pedigree harbored the mutations either 11778G > A, 14484T > C or 3460G > A, respectively, accounting for 66.7%. The mutation 11778G > A in these patients was homoplasmic and prevalent (55.5%, 10 of 18 cases). The mutations 3460G > A and 3394T > C were found to co-exist in one LHON case. The mutation 13708G > A appeared in one LHON pedigree. Smaller amount of sampling and reaction volume, easier target preparation, fast and high-throughput were the main advantages of the biochip over direct DNA sequencing and pyrosequencing. Our findings suggested that primary mutations of 11778G > A, 14484T > C or 3460G > A are main variants of mtDNA gene leading to LHON in China. The biochip would easily be implemented in clinical diagnosis.

A Simple Oligonucleotide Biochip Capable of Rapidly Detecting Known Mitochondrial DNA Mutations in Chinese Patients with Leber’S Hereditary Optic Neuropathy (LHON)

Leber's hereditary optic neuropathy (LHON) is a maternally transmitted disease. Clinically, no efficient assay protocols have been available. In this study, we aimed to develop an oligonucleotide biochip specialized for detection of known base substitution mutations in mitochondrial DNA causing LHON and to investigate frequencies of LHON relevant variants in Anhui region of China. Thirty-two pairs of oligonucleotide probes matched with the mutations potentially linked to LHON were covalently immobilized. Cy5-lablled targets were amplified from blood DNA samples by a multiplex PCR method. Two kinds of primary mutations 11778 G > A and 14484 T > C from six confirmed LHON patients were interrogated to validate this biochip format. Further, fourteen Chinese LHON pedigrees and twenty-five unrelated healthy individuals were investigated by the LHON biochip, direct sequencing and pyrosequencing, respectively. The biochip was found to be able efficiently to discriminate homoplasmic and heteroplasmic mtDNA mutations in LHON. Biochip analysis revealed that twelve of eighteen LHON symptomatic cases from the 14 Chinese pedigree harbored the mutations either 11778G > A, 14484T > C or 3460G > A, respectively, accounting for 66.7%. The mutation 11778G > A in these patients was homoplasmic and prevalent (55.5%, 10 of 18 cases). The mutations 3460G > A and 3394T > C were found to co-exist in one LHON case. The mutation 13708G > A appeared in one LHON pedigree. Smaller amount of sampling and reaction volume, easier target preparation, fast and high-throughput were the main advantages of the biochip over direct DNA sequencing and pyrosequencing. Our findings suggested that primary mutations of 11778G > A, 14484T > C or 3460G > A are main variants of mtDNA gene leading to LHON in China. The biochip would easily be implemented in clinical diagnosis.

Strategies for preparation of oligonucleotide biochips and their applications

In the last few years, DNA-microarray technology has emerged as a powerful tool for gene discovery, detection of mutations, and mapping. Here, we have developed a simple and efficient single-step method for immobilization of phosphoryl-, mercaptoalkyl-, thiophosphoryl- and aminooxyalkylated oligonucleotides onto an epoxylated glass surface. It resulted in higher immobilization and hybridization efficiency in comparison to those obtained with the standard method. The probes immobilized were found to be stable over a range of temperature and pH, suggesting that the chemistry could be used in integrated PCR/microarray devices. Also the immobilized spots were found to be of superior quality in terms of spot morphology, homogeneity and signal reproducibility. The constructed microarrays were successfully used for the discrimination of nucleotide mismatches and detection of bacterial diseases.

Development of a DNA biochip for detection of known mtDNA mutations associated with MELAS and MERRF syndromes

We developed an oligonucleotide biochip for synchronous multiplex detection of 31 known mitochondrial DNA mutations associated with MELAS (Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes) and MERRF (Myoclonic epilepsy with ragged red fibers). Allele-specific oligonucleotide probes were covalently immobilized on aldehyde modified glass slides, and then hybridized with Cy5-labled DNA fragments amplified from sample DNAs by a multiplex asymmetric PCR (MAP) method. Five patients with MELAS, 5 patients with MERRF and 20 healthy controls were investigated using the oligonucleotide biochip. The results showed that all the cases with MELAS had an A3243G mutation in the MT-TL1 gene. In the MERRF group, 4 cases were found to be an A8344G mutation and 1 case was a T8356C mutation, and both mutations were in the MT-TK gene. In the healthy controls, none of the 31 related mutations was found. The results of the DNA biochip were consistent with those by DNA sequencing. Clearly, the DNA biochip combined with MAP method would become a valuable tool in multiplex detecting of the point mutations in mtDNA leading to MELAS and/or MERRF syndrome. Moreover, this biochip format could be modified to extend to the screening scope of SNPs for any other human mitochondrial diseases.

Expression Profiles in Chronic Lymphoid Leukemia by Hematochip Analysis.

Aim: The gene expression analysis system is a good tool to identify biomarkers related to prognosis or response predictors of therapy in haematological malignancies. Low density microarrays are an useful technology to obtain genetic expression profiles in different neoplastic disorders. Recently we have designed an oligonucleotide biochip (Hematochip, Alvarez et al, Clin Chem 2007; 53:259) useful for study peripheral blood in hematological malignancies. Chronic Lymphoid Leukemia (CLL) is a frequent clonal lymphocyte disorder characterized by clinical diversity and that have known prognosis markers but a more accurate subclassification could be required. Our objective was to define CLL subgroups with differential genetic profile expression. Patients andMethods: Analytical, prospective study in 49 consecutive CLL patients diagnosed since January 2003 to December 2004 in the Hematology Department of Miguel Servet University Hospital. Zaragoza. Spain. At diagnosis demographic, clinical stage, analytical, immunophenotype expression, genetic aberrations and mutational status of IgVH were collected at electronically data base. Simultaneously, peripheral blood from 20 healthy donors and all CLL patients were collected in PAXgene tubes (PreAnalytix). cDNA was generated from total RNA and double-strand cDNA was used as a template to generate biotinylated cRNA by in vitro transcription using the MEGA-script T7 High Yield Transcription kit (Ambion). Fragmented cRNA was denatured and hybridized using a Ventana Discovery Station (Ventana Medical Systems). Arrays were stained with Cy3-conjugated streptavidin (Amersham Biosciences) and scanned using a ScanArray 4000 scanner (Perkin-Elmer). The data were treated and analyzed by GeneSpring clusters method. The expression profiles were compared and stratified with clinical stability or progression, gender, ZAP 70 expression, associated neoplasia, time free of therapy and response or not to Fludarabine.Results: The Hematochip has identified 9 probes obtained with filtered data and statistical significance (p<0.01), related to stable or progressive disease. The results had been validated by RT-PCR.Comments: the study of genetic profiles by Hematochip could be a useful analysis to predict the stability or progression of CLL patients at diagnosis. Further studies analyzing more cases will be necessary in order to validate these results. This work has been partially sponsorized by grants: Mutua Madrilena del Automovil, Fundacion para el Estudio de la Hematologia y Hemoterapia de Aragon and I+CS.

Microchips in the laboratory of A.D. Mirzabekov: 1988–2007

The article reviews the last period of A. D. Mirzabekov's scientific career. During this time, gel-based biochips were invented, studied, and introduced to practical applications in his laboratory. This work began at the early stages of the "Human Genome" project and is continuing today, including recent development of diagnostic oligonucleotide and protein biochips. This research is discussed in the context of the worldwide development of microarray technologies.

Detection of gyrA and parC Mutations Associated with Ciprofloxacin Resistance in Neisseria gonorrhoeae by Use of Oligonucleotide Biochip Technology

An oligonucleotide biochip that specifically detects point mutations in the gyrA and parC genes of Neisseria gonorrhoeae was designed and subsequently evaluated with 87 untreated clinical specimens. The susceptibilities of the N. gonorrhoeae strains were tested to determine the prevalence of ciprofloxacin-resistant strains in Anhui Province, People's Republic of China. Conventional DNA sequencing was also performed to identify mutations in gyrA and parC and to confirm the biochip data. The study demonstrates that all of the point mutations in the gyrA and parC genes of N. gonorrhoeae were easily discriminated by use of the oligonucleotide biochip. Fifteen different alteration patterns involved in the formation of ciprofloxacin resistance were identified by the biochip assay. Double mutations in both Ser91 and Asp95 of the GyrA protein were seen in all nonsensitive isolates. Double mutations in Ser91 and Asp95 of GyrA plus mutation of Glu91 or Ser87 of the ParC protein lead to significant high-level resistance to ciprofloxacin in N. gonorrhoeae isolates. The results obtained by use of the oligonucleotide biochip were identical to those obtained by use of DNA sequencing. In conclusion, the oligonucleotide biochip technology has potential utility for the rapid and reliable identification of point mutations in the drug resistance genes of N. gonorrhoeae.