RESUMO
DNA hybridization phenomena occurring on solid supports are not understood as clearly as aqueous phase hybridizations and mathematical models cannot predict some empirically obtained results. Ongoing research has identified important parameters but remains incomplete to accurately account for all interactions. It has previously been shown that the length of the overhanging (dangling) end of the target DNA strand following hybridization to the capture probe is correlated to interactions with the complementary strand in solution which can result in unbinding of the target and its release from the surface. We have developed an instrument for real-time monitoring of DNA hybridization on spherical particles functionalized with oligonucleotide capture probes and arranged in the form of a tightly packed monolayer bead bed inside a microfluidic cartridge. The instrument is equipped with a pneumatic module to mediate displacement of fluid on the cartridge. We compared this system to both conventional (passive) and centrifugally-driven (active) microfluidic microarray hybridization on glass slides to establish performance levels for the detection of single nucleotide polymorphisms. The system was also used to study the effect of the dangling end's length in real-time when the immobilized target DNA is exposed to the complementary strand in solution. Our findings indicate that increasing the length of the dangling end leads to desorption of target amplicons from bead-bound capture probes at a rate approaching that of the initial hybridization process. Finally, bead bed hybridization was performed with Streptococcus agalactiae cfb gene amplicons obtained from randomized clinical samples, which allowed for identification of group B streptococci within 5-15 min. The methodology presented here is useful for investigating competitive hybridization mechanisms on solid supports and to rapidly validate the suitability of microarray capture probes.
Assuntos
DNA , Microfluídica , DNA/genética , Sondas de DNA/genética , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos/genéticaRESUMO
Simple and fast methods for the detection of target genes with single-nucleotide specificity could open up genetic research and diagnostics beyond laboratory settings. We recently reported a biosensor for the electronic detection of unamplified target genes using liquid-gated graphene field-effect transistors employing an RNA-guided catalytically deactivated CRISPR-associated protein 9 (Cas9) anchored to a graphene monolayer. Here, using unamplified genomic samples from patients and by measuring multiple types of electrical response, we show that the biosensors can discriminate within one hour between wild-type and homozygous mutant alleles differing by a single nucleotide. We also show that biosensors using a guide RNA-Cas9 orthologue complex targeting genes within the protospacer-adjacent motif discriminated between homozygous and heterozygous DNA samples from patients with sickle cell disease, and that the biosensors can also be used to rapidly screen for guide RNA-Cas9 complexes that maximize gene-targeting efficiency.
Assuntos
Técnicas Biossensoriais/métodos , Proteína 9 Associada à CRISPR/metabolismo , DNA/genética , Polimorfismo de Nucleotídeo Único , Anemia Falciforme/genética , Anemia Falciforme/patologia , Técnicas Biossensoriais/instrumentação , Proteína 9 Associada à CRISPR/química , DNA/metabolismo , Genoma Humano , Grafite/química , Heterozigoto , Homozigoto , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Superóxido Dismutase-1/genética , Transistores EletrônicosRESUMO
Most methods for the detection of nucleic acids require many reagents and expensive and bulky instrumentation. Here, we report the development and testing of a graphene-based field-effect transistor that uses clustered regularly interspaced short palindromic repeats (CRISPR) technology to enable the digital detection of a target sequence within intact genomic material. Termed CRISPR-Chip, the biosensor uses the gene-targeting capacity of catalytically deactivated CRISPR-associated protein 9 (Cas9) complexed with a specific single-guide RNA and immobilized on the transistor to yield a label-free nucleic-acid-testing device whose output signal can be measured with a simple handheld reader. We used CRISPR-Chip to analyse DNA samples collected from HEK293T cell lines expressing blue fluorescent protein, and clinical samples of DNA with two distinct mutations at exons commonly deleted in individuals with Duchenne muscular dystrophy. In the presence of genomic DNA containing the target gene, CRISPR-Chip generates, within 15 min, with a sensitivity of 1.7 fM and without the need for amplification, a significant enhancement in output signal relative to samples lacking the target sequence. CRISPR-Chip expands the applications of CRISPR-Cas9 technology to the on-chip electrical detection of nucleic acids.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Grafite/química , Proteínas Imobilizadas/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Transistores Eletrônicos , DNA/genética , Distrofina/genética , Éxons/genética , Genoma , Células HEK293 , Humanos , Masculino , Distrofia Muscular de Duchenne/genética , Mutação/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
We present an all-thermoplastic integrated sample-to-answer centrifugal microfluidic Lab-on-Disc system (LoD) for nucleic acid analysis. The proposed CD system and engineered platform were employed for analysis of Bacillus atrophaeus subsp. globigii spores. The complete assay comprised cellular lysis, polymerase chain reaction (PCR) amplification, amplicon digestion, and microarray hybridization on a plastic support. The fluidic robustness and operating efficiency of the assay were ensured through analytical optimization of microfluidic tools enabling beneficial implementation of capillary valves and accurate control of all flow timing procedures. The assay reliability was further improved through the development of two novel microfluidic strategies for reagents mixing and flow delay on the CD platform. In order to bridge the gap between the proof-of-concept LoD and production prototype demonstration, low-cost thermoplastic elastomer (TPE) was selected as the material for CD fabrication and assembly, allowing the use of both, high quality hot-embossing and injection molding processes. Additionally, the low-temperature and pressure-free assembly and bonding properties of TPE material offer a pertinent solution for simple and efficient loading and storage of reagents and other on-board components. This feature was demonstrated through integration and conditioning of microbeads, magnetic discs, dried DNA buffer reagents and spotted DNA array inserts. Furthermore, all microfluidic functions and plastic parts were designed according to the current injection mold-making knowledge for industrialization purposes. Therefore, the current work highlights a seamless strategy that promotes a feasible path for the transfer from prototype toward realistic industrialization. This work aims to establish the full potential for TPE-based centrifugal system as a mainstream microfluidic diagnostic platform for clinical diagnosis, water and food safety, and other molecular diagnostic applications.
Assuntos
DNA Bacteriano/análise , Elastômeros/química , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase , Bacillus subtilis/genética , Carbocianinas/química , Técnicas Analíticas Microfluídicas/instrumentação , Análise de Sequência com Séries de Oligonucleotídeos/instrumentaçãoRESUMO
The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have high complexity and cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotide sequence-dependent segment and a unique "target sequence-independent" 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.
Assuntos
DNA Viral/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Oligonucleotídeos/química , Reação em Cadeia da Polimerase/métodos , Fluorescência , Humanos , Vírus da Influenza A/genética , Vírus da Influenza B/genética , Sondas de Oligonucleotídeos/química , Vírus Sinciciais Respiratórios/genéticaRESUMO
We report a new electrochemical amplification strategy for an ultrasensitive electrochemical detection of DNA sequences using aggregates composed of a water-soluble, ferrocene-functionalized polythiophene. A two-step hybridization is performed at one addressing surface with PNA capture probes whereas the electrochemical detection is done on an electrode nearby. Specific and quantitative detection of DNA targets with a detection limit of 4 × 10(-16) M (about 4 zeptomoles or about 2500 copies of oligonucleotides) was achieved.
Assuntos
Técnicas Biossensoriais , Cátions/química , DNA/análise , Eletroquímica , Compostos Ferrosos/química , Ácidos Nucleicos Peptídicos/química , Polímeros/química , Tiofenos/química , DNA/genética , Sondas de DNA , Eletrodos , Ouro/química , Metalocenos , Nanoestruturas , Sensibilidade e EspecificidadeRESUMO
A novel, centrifugal disk-based micro-total analysis system (µTAS) for low cost and high throughput semi-automated immunoassay processing was developed. A key innovation in the disposable immunoassay disk design is in a fluidic structure that enables very efficient micro-mixing based on a reciprocating mechanism in which centrifugal acceleration acting upon a liquid element first generates and stores pneumatic energy that is then released by a reduction of the centrifugal acceleration, resulting in a reversal of direction of flow of the liquid. Through an alternating sequence of high and low centrifugal acceleration, the system reciprocates the flow of liquid within the disk to maximize incubation/hybridization efficiency between antibodies and antigen macromolecules during the incubation/hybridization stage of the assay. The described reciprocating mechanism results in a reduction in processing time and reagent consumption by one order of magnitude.
Assuntos
Centrifugação/instrumentação , Imunoensaio/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Antígenos de Bactérias/análise , Antígenos de Bactérias/imunologia , Burkholderia , Humanos , Imunoglobulina G/imunologia , Indicadores e Reagentes/química , Fatores de TempoRESUMO
The applications of microfluidic technologies in medical diagnostics continue to increase, particularly in the field of nucleic acid diagnostics. While much attention has been focused on the development of nucleic acid amplification and detection platforms, sample preparation is often taken for granted or ignored all together. Specifically, little or no consideration is paid to the development of microfluidic systems that efficiently extract nucleic acids from biological samples. Here, a centrifugal microfluidic platform for mechanical sample lysis and homogenization is presented. The system performs sample lysis through a magnetically actuated bead-beating system followed by a centrifugal clarification step. The supernatant is then transferred for extraction using a unique siphon. Several other new microfluidic functions are implemented on this centrifugal platform as well, including sample distribution, a unique hydraulic capillary valve, and self-venting. Additionally, the improved system has features with a small footprint designed specifically for integration with further downstream processing steps. Biological validation of the platform is performed using Bacillus subtilis spores and clinical samples (nasopharyngeal aspirates) for respiratory virus detection. The platform was found to be as efficient as in-tube bead-beating lysis and homogenization for nucleic acid extraction, and capable of processing 4 samples in batch to near PCR-ready products in under 6 min.
Assuntos
Fracionamento Celular/instrumentação , Centrifugação/instrumentação , Técnicas Analíticas Microfluídicas/instrumentação , Ácidos Nucleicos/isolamento & purificação , Manejo de Espécimes/instrumentação , Fracionamento Químico , Desenho Assistido por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Commonly used internal controls (ICs) to monitor the efficiency of nucleic acid testing (NAT) assays do not allow verification of nucleic acid extraction efficiency. Since microbial cells are often difficult to lyse, it is important to ensure that nucleic acids are efficiently extracted from any target organism. For this purpose, we developed a cellular IC based on the use of nonpathogenic Bacillus spores. Purified Bacillus atrophaeus subsp. globigii (referred to hereafter as simply B. atrophaeus) spores were added to vaginal and anal samples, which were then subjected to rapid DNA extraction and subsequent PCR amplification. The proof of concept of this cellular IC was made through the use of both manual and automated DNA extraction methods, using vaginal or anal samples spiked with B. atrophaeus spores, combined with a multiplex real-time PCR assay for the specific detection of group B streptococci (GBS) and B. atrophaeus. The performance of the cellular IC was compared to that of a standard IC plasmid added to PCRs. Approximately 500 B. atrophaeus spores per PCR was found to be optimal since this did not interfere significantly with GBS detection for either DNA extraction method and yielded reproducible amplification and/or detection of B. atrophaeus genomic DNA serving as an IC template. Performance of the cellular IC was comparable to that of the standard IC. This novel IC system using nonpathogenic and hard-to-lyse B. atrophaeus spores allowed validation of both the DNA extraction procedure and the amplification and detection process. Use of a spore-based control also provides a universal control for microbial cell lysis.
Assuntos
Bacillus/genética , DNA Bacteriano/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/normas , Esporos Bacterianos/genética , Infecções Estreptocócicas/diagnóstico , Streptococcus agalactiae/isolamento & purificação , Canal Anal/microbiologia , Bacillus/fisiologia , Bactérias/genética , Bactérias/isolamento & purificação , DNA Bacteriano/genética , Feminino , Humanos , Reação em Cadeia da Polimerase/métodos , Padrões de Referência , Reprodutibilidade dos Testes , Streptococcus agalactiae/genética , Vagina/microbiologiaRESUMO
Fabrication of microarray devices using traditional glass slides is not easily adaptable to integration into microfluidic systems. There is thus a need for the development of polymeric materials showing a high hybridization signal-to-background ratio, enabling sensitive detection of microbial pathogens. We have developed such plastic supports suitable for highly sensitive DNA microarray hybridizations. The proof of concept of this microarray technology was done through the detection of four human respiratory viruses that were amplified and labeled with a fluorescent dye via a sensitive reverse transcriptase PCR (RT-PCR) assay. The performance of the microarray hybridization with plastic supports made of PMMA [poly(methylmethacrylate)]-VSUVT or Zeonor 1060R was compared to that with high-quality glass slide microarrays by using both passive and microfluidic hybridization systems. Specific hybridization signal-to-background ratios comparable to that obtained with high-quality commercial glass slides were achieved with both polymeric substrates. Microarray hybridizations demonstrated an analytical sensitivity equivalent to approximately 100 viral genome copies per RT-PCR, which is at least 100-fold higher than the sensitivities of previously reported DNA hybridizations on plastic supports. Testing of these plastic polymers using a microfluidic microarray hybridization platform also showed results that were comparable to those with glass supports. In conclusion, PMMA-VSUVT and Zeonor 1060R are both suitable for highly sensitive microarray hybridizations.
Assuntos
Análise em Microsséries/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Plásticos , Polímeros , Vírus/isolamento & purificação , Humanos , RNA Viral/análise , RNA Viral/genética , Sensibilidade e Especificidade , Vírus/genéticaRESUMO
A series of vancomycin-modified nanoparticles were developed and employed in magnetic confinement assays to isolate a variety of Gram-positive and Gram-negative bacteria from aqueous solution. We determined that the orientation/architecture of vancomycin on the surface of the nanoparticles and the overall surface coverage is critical in mediating fast and effective interactions between the nanoparticle and the pathogen cell wall surface and only one orientation/architecture in a series of modified nanoparticles leads to the efficient and reproducible capture of several important pathogenic bacteria. Interestingly, as the nanoparticles increase in diameter (from approximately 50 to 2800 nm), it is necessary to incorporate a long linker between the nanoparticle surface and the vancomycin moiety in order for the surface bound probe to efficiently confine Gram-positive bacteria. Finally, we also determined that the time required for efficient labeling and subsequent magnetic confinement significantly decreases as the size of the nanoparticle and the vancomycin surface coverage on the nanoparticle increases. As disease detection technologies transition to "lab-on-a-chip" based platforms it is necessary to develop strategies to effectively and inexpensively preconcentrate cells from large volume to volumes more amenable to these types of microfluidic devices. These small molecule-modified superparamagnetic nanoparticles can provide a means by which this can be accomplished.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Nanopartículas/administração & dosagem , Vancomicina/administração & dosagem , Vancomicina/química , Antibacterianos/administração & dosagem , Antibacterianos/química , Portadores de Fármacos/química , Bactérias Gram-Negativas/isolamento & purificação , Bactérias Gram-Positivas/isolamento & purificação , Teste de Materiais , Nanomedicina/métodos , Nanopartículas/químicaAssuntos
DNA/química , Exodesoxirribonucleases/química , Candida/genética , Carbocianinas , DNA/genética , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Fúngico/química , DNA Fúngico/genética , Corantes Fluorescentes , Listeria monocytogenes/genética , Técnicas Microbiológicas , Neisseria meningitidis/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo ÚnicoRESUMO
BACKGROUND: Group B streptococci (GBS) are a leading cause of sepsis and meningitis in newborns. We previously developed a rapid diagnostic system for GBS detection from vaginal/anal samples obtained from pregnant women during delivery. To facilitate the adaptation of this method for point-of-care testing, we have developed a specific and efficient GBS DNA capture method that is compatible with both PCR and nonamplification detection technologies. METHODS: Superparamagnetic beads were functionalized with oligonucleotide capture probes of different lengths and used to capture GBS genomic DNA (gDNA). A rapid extraction procedure was used to provide DNA from GBS cultures or vaginal/anal samples with added GBS. Hybridization reactions consisting of functionalized beads and target DNA in 30 muL of hybridization buffer were performed for 1 h at room temperature, followed by washing and resuspension in water. Captured DNA was then detected using quantitative PCR. RESULTS: A 25-mer capture probe allowed detection of 1000 genome copies of purified GBS DNA. The ability to detect GBS was improved by use of a 50-mer (100 copies) and a 70-mer capture probe (10 copies). Detection of approximately 1250 CFU/mL was achieved for diluted GBS broth culture and for vaginal/anal swab samples with added GBS. CONCLUSION: Oligonucleotide-functionalized superparamagnetic microbeads efficiently capture GBS gDNA from both bacterial cultures and vaginal/anal samples with added GBS. Efficiency of gDNA capture increases with oligonucleotide length. This technology could be combined with sample preparation and detection technologies in a microfluidic system to allow point-of-care testing for GBS.
Assuntos
Canal Anal/microbiologia , DNA Bacteriano/análise , Genoma Bacteriano , Streptococcus agalactiae/genética , Vagina/microbiologia , Técnicas Bacteriológicas , Parto Obstétrico , Feminino , Humanos , Magnetismo , Microesferas , Sistemas Automatizados de Assistência Junto ao Leito , Gravidez , Streptococcus agalactiae/isolamento & purificaçãoRESUMO
Microarrays have become one of the most convenient tools for high throughput screening, supporting major advances in genomics and proteomics. Other important applications can be found in medical diagnostics, detection of biothreats, drug discovery, etc. Integration of microarrays with microfluidic devices can be highly advantageous in terms of portability, shorter analysis time and lower consumption of expensive biological analytes. Since fabrication of microfluidic devices using traditional materials such as glass is rather expensive, there is great interest in employing polymeric materials as a low cost alternative that is suitable for mass production. A number of commercially available plastic materials were reviewed for this purpose and poly(methylmethacrylate) Zeonor 1060R and Zeonex E48R were identified as promising candidates, for which methods for surface modification and covalent immobilization of DNA oligonucleotides were developed. In addition, we present proof-of-concept plastic-based microarrays with and without integration with microfluidics.
Assuntos
Técnicas Analíticas Microfluídicas , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Plásticos/química , Análise Serial de Proteínas/instrumentação , Alcenos/química , Carbono/química , DNA/química , Desenho de Equipamento , Manufaturas , Microfluídica , Eletricidade Estática , Propriedades de SuperfícieRESUMO
A low-cost, disposable card for rapid polymerase chain reaction (PCR) was developed in this work. Commercially available, adhesive-coated aluminum foils and polypropylene films were laminated with structured polycarbonate films to form microreactors in a card format. Ice valves [1] were employed to seal the reaction chambers during thermal cycling and a Peltier-based thermal cycler was configured for rapid thermal cycling and ice valve actuation. Numerical modeling was conducted to optimize the design of the PCR reactor and investigate the thermal gradient in the reaction chamber in the direction of sample thickness. The PCR reactor was experimentally characterized by using thin foil thermocouples and validated by a successful amplification of 10 copy of E. coli tuf gene in 27 min.
Assuntos
Equipamentos Descartáveis/economia , Reação em Cadeia da Polimerase/instrumentação , Desenho de Equipamento , Escherichia coli/genética , Miniaturização , Modelos Teóricos , Reação em Cadeia da Polimerase/economia , Reação em Cadeia da Polimerase/métodos , TemperaturaRESUMO
The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.
Assuntos
Benchmarking/métodos , Sondas de DNA/genética , Hibridização In Situ/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Análise de Sequência de DNA/métodos , Hibridização In Situ/normas , Análise de Sequência com Séries de Oligonucleotídeos/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/normas , Estatística como AssuntoRESUMO
BACKGROUND: Current hybridization protocols on microarrays are slow and need skilled personnel. Microfluidics is an emerging science that enables the processing of minute volumes of liquids to perform chemical, biochemical, or enzymatic analyzes. The merging of microfluidics and microarray technologies constitutes an elegant solution that will automate and speed up microarray hybridization. METHODS: We developed a microfluidic flow cell consisting of a network of chambers and channels molded into a polydimethylsiloxane substrate. The substrate was aligned and reversibly bound to the microarray printed on a standard glass slide to form a functional microfluidic unit. The microfluidic units were placed on an engraved, disc-shaped support fixed on a rotational device. Centrifugal forces drove the sample and buffers directly onto the microarray surface. RESULTS: This microfluidic system increased the hybridization signal by approximately 10fold compared with a passive system that made use of 10 times more sample. By means of a 15-min automated hybridization process, performed at room temperature, we demonstrated the discrimination of 4 clinically relevant Staphylococcus species that differ by as little as a single-nucleotide polymorphism. This process included hybridization, washing, rinsing, and drying steps and did not require any purification of target nucleic acids. This platform was sensitive enough to detect 10 PCR-amplified bacterial genomes. CONCLUSION: This removable microfluidic system for performing microarray hybridization on glass slides is promising for molecular diagnostics and gene profiling.
Assuntos
DNA/química , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Dimetilpolisiloxanos/química , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Sensibilidade e Especificidade , Especificidade da Espécie , Staphylococcus/classificação , Staphylococcus/genética , Propriedades de SuperfícieRESUMO
BACKGROUND: Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization. RESULTS: Using surface-bound peptide nucleic acids (PNA) probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray. CONCLUSION: This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.
Assuntos
Biotecnologia/métodos , DNA/análise , Corantes Fluorescentes/farmacologia , Sondas de Ácido Nucleico/farmacologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ácidos Nucleicos Peptídicos/química , Eletricidade Estática , Sequência de Bases , Técnicas Biossensoriais/métodos , Cátions , DNA/química , Eletroquímica/métodos , Técnicas Genéticas , Dados de Sequência Molecular , Mutação , Hibridização de Ácido Nucleico , Ácidos Nucleicos/análise , Ácidos Nucleicos/química , Polímeros/química , Espectrometria de Fluorescência/métodos , Tiofenos/químicaRESUMO
In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes gamma-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance.