RESUMO
We describe a novel method for rapidly identifying and distinguishing between different DNA sequences using short tandem repeat (STR) analysis and DNA microarrays. The method can be used to deduce identity, length, and number of STRs of the target molecule. We refer to this technique as the "variable-length probe array" method for STR profiling (VLPA). The method involves hybridization of the unknown STR target sequence to a DNA microarray displaying complementary probes that vary in length to cover the range of possible STRs. A post-hybridization enzymatic digestion of the DNA hybrids is then used to selectively remove labeled single-stranded regions of DNA from the microarray surface. The number of repeats in the unknown target is then deduced based on the pattern of target DNA that remains hybridized to the array. This DNA profiling technique is useful for performing forensic analysis to uniquely identify individual humans or other species.
Assuntos
Impressões Digitais de DNA/métodos , DNA/análise , Análise de Sequência com Séries de Oligonucleotídeos , Sequências de Repetição em Tandem , Humanos , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Análise de Sequência de DNA , Endonucleases Específicas para DNA e RNA de Cadeia SimplesRESUMO
The 61 CTSA Consortium sites are home to valuable programs and infrastructure supporting translational science and all are charged with ensuring that such investments translate quickly to improved clinical care. Catalog of Assets for Translational and Clinical Health Research (CATCHR) is the Consortium's effort to collect and make available information on programs and resources to maximize efficiency and facilitate collaborations. By capturing information on a broad range of assets supporting the entire clinical and translational research spectrum, CATCHR aims to provide the necessary infrastructure and processes to establish and maintain an open-access, searchable database of consortium resources to support multisite clinical and translational research studies. Data are collected using rigorous, defined methods, with the resulting information made visible through an integrated, searchable Web-based tool. Additional easy-to-use Web tools assist resource owners in validating and updating resource information over time. In this paper, we discuss the design and scope of the project, data collection methods, current results, and future plans for development and sustainability. With increasing pressure on research programs to avoid redundancy, CATCHR aims to make available information on programs and core facilities to maximize efficient use of resources.
Assuntos
Catálogos como Assunto , Comportamento Cooperativo , Pesquisa sobre Serviços de Saúde , Pesquisa Translacional Biomédica , Coleta de Dados , Ensaios de Triagem em Larga Escala , Humanos , Internet , Reprodutibilidade dos Testes , Interface Usuário-ComputadorRESUMO
STR analysis is commonly used in forensic and genetic studies. STRs are currently discriminated based on size, primarily by gel- and column-based approaches. Hybridization-based approaches have the potential to allow high-throughput analysis of STRs; however, development of such approaches has been limited by the difficulty in discriminating between STRs of similar length. We have recently described several innovations to enable STR analysis using an array-based hybridization approach for high- throughput STR analysis. Here we extend that approach by incorporating the array into microspheres and adding a discriminatory branch migration displacement step. This microsphere-based platform uses Luminex xMAP technology and improves the sensitivity, selectivity, and speed of the assay. We demonstrate the feasibility, speed, and reliability of the assay for STR detection by correctly analyzing two STR loci in 20 forensic DNA samples of known STR type. The multiplex, bead-based approach provides a high-throughput and more portable STR analysis.
Assuntos
Repetições de Microssatélites , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Humanos , Microesferas , Hibridização de Ácido Nucleico/métodos , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Espectrometria de Fluorescência/métodos , SuspensõesRESUMO
Ion current rectification with quartz nanopipette electrodes was investigated through the control of the surface charge. The presence and absence of a positively charged poly-l-lysine (PLL) coating resulted in the rectified current with opposite polarity. The results agreed with the theories developed for current-rectifying conical nanopores, suggesting the similar underlying mechanism among asymmetric nanostructure in general. This surface condition dependence can be used as the fundamental principle of multi-purpose real-time in vivo biosensors.
Assuntos
Nanotubos/química , Polilisina/química , Quartzo/química , Condutividade Elétrica , Eletrodos , Teste de Materiais , Nanotecnologia/instrumentação , Nanotecnologia/métodos , Tamanho da Partícula , Sensibilidade e Especificidade , Propriedades de SuperfícieRESUMO
Rapid, sequence-specific DNA detection is essential for applications in medical diagnostics and genetic screening. Electrical biosensors that use immobilized nucleic acids are especially promising in these applications because of their potential for miniaturization and automation. Current DNA detection methods based on sequencing by synthesis rely on optical readouts; however, a direct electrical detection method for this technique is not available. We report here an approach for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface charge perturbation. We discovered that incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface charge perturbation. This event can be detected as a transient current by a voltage-clamp amplifier. Based on current understanding of polarizable interfaces, we propose that the electrode detects proton removal from the 3'-hydroxyl group of the DNA molecule during phosphodiester bond formation.
Assuntos
Técnicas Biossensoriais/métodos , DNA/biossíntese , Sequência de Bases , Técnicas Biossensoriais/instrumentação , DNA/química , Eletroquímica , Eletrodos , Ouro , Cinética , Propriedades de SuperfícieRESUMO
Single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The nanopipette detection technique can provide not only tools for detection and identification of single DNA and protein molecules but also deeper insight and understanding of stochastic interactions of various biomolecules with their environment.