DNA microarray Dotaz Zobrazit nápovědu
DNA microarrays (collections of DNA probes arranged on a shared base) have recently enlarged the spectrum of commercially available laboratory-ready kits in molecular biology. They are powerful new tools for the investigation of global changes in gene expression profiles in cells and tissues. Their assembly process is automatized and the DNA microarrays are further miniaturized. The DNA microarrays are used in search for various specific genes (e.g. connected with an infectious agent) or in gene polymorphism and expression analysis. They will be widely used to investigate expression of various genes connected with various diseases in order to find causes of these diseases and to enable their accurate treatment. Since the DNA microarray assembly technology has been based on methods widely used in the semiconductor industry, we can expect a rapid onset of the routine use of this revolutionary device.
- MeSH
- biotechnologie MeSH
- DNA genetika MeSH
- exprese genu MeSH
- lidé MeSH
- sekvence nukleotidů MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů * přístrojové vybavení metody trendy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- DNA MeSH
DNA microarray assay has become a useful tool for gene expression studies. Less frequent is its application to detection of viruses or diagnostics of virus diseases. Here we show design of a microscope slide-based microarray assay for simultaneous identification of several potato viruses. Different primer pairs were designed or adopted to obtain specific amplicons from six potato viruses: Potato virus A (PVA), Potato virus S (PVS), Potato virus X (PVX), Potato virus Y (PVY), Potato mop-top virus (PMTV) and Potato leaf-roll virus (PLRV). Purified viral DNA probes were spotted on a microscope slide coated with poly-L-lysine. The same primers were used for preparation of fluorochrome-labeled targets. The latter were denatured and hybridized on the microarray slide (chip). An example of simultaneous assay of two pathogens is given and possibilities of practical application of this type of assay are discussed.
- MeSH
- DNA primery genetika MeSH
- DNA virů analýza izolace a purifikace MeSH
- nemoci rostlin virologie MeSH
- Potexvirus genetika izolace a purifikace MeSH
- Potyvirus genetika izolace a purifikace MeSH
- rostlinné viry genetika izolace a purifikace MeSH
- sekvenční analýza DNA MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody statistika a číselné údaje MeSH
- sekvenční homologie MeSH
- senzitivita a specificita MeSH
- Solanum tuberosum virologie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA primery MeSH
- DNA virů MeSH
The combination of microarray technologies with microfluidic sample delivery and real-time detection methods has the capability to simultaneously monitor 10-1000 s of biomolecular interactions in a single experiment. Despite the benefits that microfluidic systems provide, they typically operate in the laminar flow regime under mass transfer limitations, where large analyte depletion layers act as a resistance to analyte capture. By locally stirring the fluid and delivering fresh analyte to the capture spot, the use of passive mixing structures in a microarray environment can reduce the negative effects of these depletion layers and enhance the sensor performance. Despite their large potential, little attention has been given to the integration of these mixing structures in microarray sensing environments. In this study, we use passive mixing structures to enhance the mass transfer of analyte to a capture spot within a microfluidic flow cell. Using numerical methods, different structure shapes and heights were evaluated as means to increase local fluid velocities, and in turn, rates of mass transfer to a capture spot. These results were verified experimentally via the real-time detection of 20-mer ssDNA for an array of microspots. Both numerical and experimental results showed that a passive mixing structure situated directly over the capture spot can significantly enhance the binding rate of analyte to the sensing surface. Moreover, we show that these structures can be used to enhance mass transfer in experiments regarding an array of capture spots. The results of this study can be applied to any experimental system using microfluidic sample delivery methods for microarray detection techniques.
- Klíčová slova
- Biosensors, Mass transfer, Microarrays, Microfluidic mixing, Microfluidics,
- MeSH
- biosenzitivní techniky přístrojové vybavení MeSH
- design vybavení MeSH
- difuze MeSH
- jednovláknová DNA analýza MeSH
- mikročipová analýza přístrojové vybavení MeSH
- mikrofluidní analytické techniky přístrojové vybavení MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- jednovláknová DNA MeSH
Metallothioneins (MTs), low molecular mass cysteine-rich proteins, which are able to bind up to 20 monovalent and up to 7 divalent heavy metal ions are widely studied due to their functions in detoxification of metals, scavenging free radicals and cells protection against the oxidative stress. It was found that the loss of the protective effects of MT leads to an escalation of pathogenic processes and carcinogenesis. The most extensive area is MTs expression for oncological applications, where the information about gene patterns is helpful for the identification biological function, resistance to drugs and creating the correct chemotherapy. In other medical applications the effect of oxidative stress to cell lines exposed to heavy metals and hydrogen peroxide is studied as well as influence of drugs and cytokines on MTs expression and MTs expression in the adipose tissue. The precise detection of low metallothionein concentrations and its isoforms is necessary to understand the connection between quantity and isoforms of MTs to size, localization and type of cancer. This information is necessary for well-timed therapy and increase the chance to survival. Microarray chips appear as good possibility for finding all information about expression of MTs genes and isoforms not only in cancer, but also in other diseases, especially diabetes, obesity, cardiovascular diseases, ageing, osteoporosis, psychiatric disorders and as the effects of toxic drugs and pollutants, which is discussed in this review.
- Klíčová slova
- Drug, Gene expression, Metallothionein, Microarray, Resistance, Tumour diseases,
- MeSH
- lidé MeSH
- metalothionein analýza genetika metabolismus MeSH
- mikročipová analýza metody MeSH
- nádory diagnóza genetika metabolismus MeSH
- oxidační stres fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- metalothionein MeSH
An oligonucleotide microarray for the detection of some fruit-tree viruses was designed and its theoretical detection limit was assessed using Cy3-labelled oligonucleotides. The real sensitivity of the microarray was compared for different kinds of fluorescently labelled targets: (a) cDNA and PCR amplified targets, (b) PCR amplified targets labelled using three different labelling methods. In the first case (a), the number of viral cDNA molecules was below the assessed detection limit of the microarray and only PCR amplified targets were detected. A second comparison (b), done on 3 selected viruses, included indirect labelling, the direct incorporation of labelled-dUTPs, and the use of Cy3-labelled primer. The targets labelled most intensively were produced by the Cy3-primer labelling (2 of 3 viruses) or by the indirect labelling method (1 of 3 viruses), the weakest signal showed targets labelled directly (all 3 viruses). The use of Cy3-primer labelling involved the simplest preparation and the lowest cost, however occasional weak cross-hybridization appeared. The indirect labelling method was of the highest specificity. The probes hybridizing near the 3-end of the targets showed the lowest intensities of fluorescent signal.
- MeSH
- falešně pozitivní reakce MeSH
- fluorescence MeSH
- mikročipová analýza metody MeSH
- nemoci rostlin virologie MeSH
- rostlinné viry izolace a purifikace MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů MeSH
- senzitivita a specificita MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
BACKGROUND: Wilson disease (WD) is a serious autosomal recessive disorder caused by mutations in ATP7B-gene which encodes a copper-specific ATPase. WD patients suffer from impaired biliary excretion of copper from organism and its' accumulation in body organs. Molecular diagnostics of WD is an important part of correct diagnosis statement. The aim of the study was to design and validate a genotyping DNA microarray which enables to analyze 87 mutations and 17 polymorphisms in ATP7B gene, simultaneously. METHODS AND RESULTS: 97 WD patients with known genotypes and 46 samples prepared by mutagenesis were tested in the first phase of chip validation. All analyzed sequence variants were detected with 100% accuracy. Samples from WD suspected patients were tested in the second phase of validation. We have analyzed 58 unrelated patients, yet. The diagnosis of WD was confirmed in 10 patients, 13 patients were heterozygous for some mutation and 35 had no mutation in ATP7B gene. Samples with one or no mutation found by microarray analysis were sequenced directly and no further causal mutation was revealed. CONCLUSIONS: Wilson chip seems to be a fast and reliable method for screening of mutations in ATP7B gene.
The success of DNA expression microarrays has been followed by applications of this technology to molecular diagnosis, mainly in the fields of biology and medicine. The experiments described below apply microarray diagnosis to agriculture. This report presents results of field tests for a DNA microarray designed to diagnose major viral potato pathogens. The assays were performed on samples that had been tested previously for the presence of viral infection by ELISA. RNA isolation methods were optimised for high sensitivity, using only 3 microg of total RNA that were reverse transcribed using random hexamers, with the resulting cDNA hybridised after labelling to an oligonucleotide array. The results obtained confirm the presence of pathogens indicated by ELISA and simultaneously reveal other viruses in the same reaction, showing that this method is appropriate for rapid detection of mixed viral infections. This observation was verified by subsequent RT-PCR and sequencing.
- MeSH
- DNA primery MeSH
- ELISA metody MeSH
- hybridizace nukleových kyselin MeSH
- komplementární DNA genetika izolace a purifikace MeSH
- nemoci rostlin virologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- RNA virová genetika izolace a purifikace MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody MeSH
- Solanum tuberosum virologie MeSH
- virologie metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- validační studie MeSH
- Názvy látek
- DNA primery MeSH
- komplementární DNA MeSH
- RNA virová MeSH
Wilson disease (WD) is an autosomal recessive inherited disorder of copper metabolism that is caused by mutations in the ATP7B gene. To date, more than 300 mutations have been described in this gene. Molecular diagnostics of WD utilizes restriction enzyme digestion, multiplex ligation-dependent probe amplification or a direct sequencing of the whole gene. To simplify and speed up the screening of ATP7B mutations, we have developed a genotyping microarray for the simultaneous detection of 87 mutations and 17 polymorphisms in the ATP7B gene based on the arrayed primer extension reaction. The patient's DNA is amplified in four multiplex polymerase chain reactions, fragmented products are annealed to arrayed primers spotted on a chip, which enables DNA polymerase extension reactions with fluorescently labeled dideoxynucleotides. The Wilson microarray was validated by screening 97 previously genetically confirmed WD patients. In total, we detected 43 mutations and 15 polymorphisms that represent a majority of the common mutations occurring in the Czech and Slovak populations. All screened sequence variants were detected with 100% accuracy. The Wilson chip appears to be a rapid, sensitive and cost-effective tool, representing the prototype of a disease chip that facilitates and speeds up the screening of potential WD patients.
- MeSH
- adenosintrifosfatasy genetika MeSH
- ATPasy transportující měď MeSH
- bodová mutace * MeSH
- detekce genetických nosičů metody MeSH
- genotyp MeSH
- hepatolentikulární degenerace diagnóza genetika MeSH
- heterozygot MeSH
- lidé MeSH
- mikročipová analýza přístrojové vybavení metody MeSH
- mutace MeSH
- mutační analýza DNA MeSH
- proteiny přenášející kationty genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- adenosintrifosfatasy MeSH
- ATP7A protein, human MeSH Prohlížeč
- ATPasy transportující měď MeSH
- proteiny přenášející kationty MeSH
Microarrays are one of the new emerging methods in plant virology currently being developed by various laboratories. In this study, a new approach is described on the detection of plant viruses using short synthetic single-stranded oligomers (40 nt) instead of PCR products as capture probes. A microchip detecting potato viruses, PVA, PVS, PVM, PVX, PVY and PLRV, in both single and mixed infections was developed and tested. The chip was also designed to distinguish between the main strains of PVY and PVS. Results of initial tests with PVY(NTN) and PVY(O) strains using several different probes for one virus are presented. Possibilities and advantages of the new oligonucleotide-based microarray approach for plant viral diagnosis are discussed.
- MeSH
- druhová specificita MeSH
- nemoci rostlin virologie MeSH
- oligonukleotidové sondy * MeSH
- oligonukleotidy * chemická syntéza chemie genetika MeSH
- rostlinné viry klasifikace genetika izolace a purifikace MeSH
- sekvenční analýza hybridizací s uspořádaným souborem oligonukleotidů metody MeSH
- senzitivita a specificita MeSH
- Solanum tuberosum virologie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- Názvy látek
- oligonukleotidové sondy * MeSH
- oligonukleotidy * MeSH
OBJECTIVE: Analysis of prenatal samples from 2015 to 2020. Comparison detection rates of clinically relevant variants by cytogenetic karyotype analysis and cytogenomic MLPA (Multiplex Ligation-Depent Probe Amplification) and microarray methods (CMA - chromosomal microarray). MATERIAL AND METHOD: 1,029 prenatal samples were analyzed by cytogenetic karyotyping (N = 1,029), cytogenomic methods - MLPA (N = 144) and CMA (N = 111). All unbalanced changes were confirmed by MLPA or CMA. RESULTS: From the analyzed set of fetuses, after subtraction of aneuploidies - 107 (10.40%, N = 1,029), 22 structural aberrations (2.39%, N = 922) - nine unbalanced changes (0.98%), 10 balanced changes (1.08%), one case of unclear mosaicism (0.09%), one case of presence of a marker chromosome (0.09%) and one case of sex discordance (0.09%) - were detected by karyotype analysis. A total of eight (7.21%, N = 111) pathological variants were detected by CMA in 255 samples with physiological karyotype indicated for cytogenomic examination. Five (3.47%, N = 144) of eight pathogenic variants were detected by MLPA method. The total capture of pathogenic variants by MLPA and CMA methods was 14 (5.14%) and 17 (6.25%) (N = 272), including confirmatory pathological karyotype testing. Detection of pathological variants in the isolated disorders group was lower than in the multiple disorders group (5.08 vs. 21.42%). CONCLUSION: A higher success rate for the detection of pathological copy number variation variants by the microarray method than by the MLPA method was confirmed.
- Klíčová slova
- CMA, MLPA, congenital developmental disorders, copy number variants,
- MeSH
- lidé MeSH
- mikročipová analýza MeSH
- mozaicismus MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- plod MeSH
- prenatální diagnóza * metody MeSH
- těhotenství MeSH
- variabilita počtu kopií segmentů DNA * MeSH
- Check Tag
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
