RESUMEN
Nucleotide analogues are used increasingly in medicine and biotechnology to effect DNA sequence change, principally via clastogenic and transcriptional effects. This article, however, discusses the use of mutagenic nucleotide analogues to improve the sequencing of recalcitrant and repetitive DNA motifs. Guidance in the technical and practical approaches that support use of this approach with different DNA sequencing technologies is provided, including for high-throughput technologies.
Asunto(s)
ADN/genética , Análisis de Secuencia de ADN , Mutación , Reacción en Cadena de la PolimerasaRESUMEN
Differential gene expression is tightly controlled by transcription factors (TFs), which bind close to target genes and interact together to activate and coregulate transcription. Bioinformatics analysis of published genome-wide gene expression data has allowed the development of comprehensive models of TFs likely to be active in particular tissues (signature TFs); however, the predicted activities of many of the TFs have not been experimentally confirmed. Here, we describe methods for the parallel analysis of the activities of more than 200 transcription factor proteins, using an advanced oligonucleotide array-based transcription factor assay (OATFA) platform, to assay TF activities in mice. The system uses a PCR-based system to translate cellular levels of target DNA-TF complex into a dye-tagged DNA signal, which is read by the developed microarray. The PCR step introduces semiquantitative amplification of the represented TF binding sequences. Experimental OATFA findings can identify many TF activities, which bioinformatics profiling does not predict. Newly identified TF activities can be confirmed by antibody-ELISA against active TFs. The PCR-based OATFA microarray analysis is a comprehensive method that can be used to reveal transcriptional systems and pathways which may function in different mammalian tissues and cells.
Asunto(s)
ADN/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Factores de Transcripción/metabolismo , Animales , RatonesRESUMEN
The abuse of antibiotic drugs during animal production remains a worldwide problem and the subsequent detection of the residues of various drugs present at low concentrations in complex biological matrices poses significant analytical challenges. The present study outlines a practical biochip assay system to identify antibiotic residues in different animal tissue extracts. The system uses a simple but efficient multiresidue sample extraction procedure to isolate the antibiotic residues which were then identified directly using high-affinity monoclonal antibodies presented in a competitive immunoassay with conjugated antibiotic hapten-chips. The hapten-chip can analyze six samples each for eight antibiotics on a single chip within 3 h. The analytical results with both artificial positive standard samples and the incurred samples show that the antibody hapten-chip system has a comparable accuracy and a similar sensitivity to a standard ultra performance liquid chromatography-mass spectrometry (MS)/MS assay. In conclusion, an effective analytical screening system based on antibody hapten-chip was developed for detecting multiple antibiotic residues from multiple samples.
Asunto(s)
Antibacterianos/análisis , Residuos de Medicamentos , Haptenos/inmunología , Inmunoensayo/métodos , Análisis de Matrices Tisulares , Animales , Anticuerpos Monoclonales , Pollos , Cromatografía Liquida , Hígado , Espectrometría de Masas , Productos de la Carne , Músculo Esquelético , PorcinosRESUMEN
Molecular systematics involves the description of the regulatory networks formed by the interconnections between active transcription factors and their target expressed genes. Here, we have determined the activities of 200 different transcription factors in six mouse tissues using an advanced mouse oligonucleotide array-based transcription factor assay (MOUSE OATFA). The transcription factor signatures from MOUSE OATFA were combined with public mRNA expression profiles to construct experimental transcriptional regulatory networks in each tissue. SRF-centered regulatory networks constructed for lung and skeletal muscle with OATFA data were confirmed by ChIP assays, and revealed examples of novel networks of expressed genes coregulated by sets of transcription factors. The combination of MOUSE OATFA with bioinformatics analysis of expressed genes provides a new paradigm for the comprehensive prediction of the transcriptional systems and their regulatory pathways in mouse.
Asunto(s)
Regulación de la Expresión Génica , Redes Reguladoras de Genes , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción , Animales , Biología Computacional , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Reproducibilidad de los Resultados , Distribución Tisular , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Detection of very small amounts of RNA based on microdissection of plant tissue is essential for modern plant biology. Mass spectroscopy technology (MassARRAY) based on Sequenomtrade mark instrumentation was adapted to determine quickly and in a high-throughput fashion (by multiplexing) the absolute amounts of mRNA of closely related soybean genes. A sensitivity of 0.1 amol (10(-19)) was achieved, representing as few as 1,000 mRNA molecules. This methodology eliminates the use of housekeeping genes as reference standards and has multiple applications for plant functional genomics, such as the monitoring of individual expression of paralogous genes at ultra-low expression levels and/or in extremely small tissue samples.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Glycine max/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Mensajero/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Genes de Plantas , Reacción en Cadena de la Polimerasa/métodos , ARN de Planta/análisis , Sensibilidad y EspecificidadRESUMEN
MOTIVATION: Despite many successes of conventional DNA sequencing methods, some DNAs remain difficult or impossible to sequence. Unsequenceable regions occur in the genomes of many biologically important organisms, including the human genome. Such regions range in length from tens to millions of bases, and may contain valuable information such as the sequences of important genes. The authors have recently developed a technique that renders a wide range of problematic DNAs amenable to sequencing. The technique is known as sequence analysis via mutagenesis (SAM). This paper presents a number of algorithms for analysing and interpreting data generated by this technique. RESULTS: The essential idea of SAM is to infer the target sequence using the sequences of mutants derived from the target. We describe three algorithms used in this process. The first algorithm predicts the number of mutants that will be required to infer the target sequence with a desired level of accuracy. The second algorithm infers the target sequence itself, using the mutant sequences. The third algorithm assigns quality values to each inferred base. The algorithms are illustrated using mutant sequences generated in the laboratory.
Asunto(s)
Algoritmos , Análisis Mutacional de ADN/métodos , Modelos Genéticos , Mutagénesis/genética , Alineación de Secuencia/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Animales , Simulación por Computador , Dictyostelium/genética , Modelos Estadísticos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Despite the success of conventional Sanger sequencing, significant regions of many genomes still present major obstacles to sequencing. Here we propose a novel approach with the potential to alleviate a wide range of sequencing difficulties. The technique involves extracting target DNA sequence from variants generated by introduction of random mutations. The introduction of mutations does not destroy original sequence information, but distributes it amongst multiple variants. Some of these variants lack problematic features of the target and are more amenable to conventional sequencing. The technique has been successfully demonstrated with mutation levels up to an average 18% base substitution and has been used to read previously intractable poly(A), AT-rich and GC-rich motifs.
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Mutagénesis/genética , Análisis de Secuencia de ADN/métodos , Secuencia Rica en At/genética , Algoritmos , Animales , Secuencia de Bases , ADN Mitocondrial/genética , ADN Protozoario/química , ADN Protozoario/genética , Dictyostelium/genética , Secuencia Rica en GC/genética , Humanos , Datos de Secuencia Molecular , ARN de Transferencia de Treonina/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
Capillary electrophoresis has advanced enormously over the last 10 yr as a tool for DNA sequencing, driven by the human and other major genome projects and by the need for rapid electrophoresis-based DNA diagnostic tests. The common need of these analyses is a platform providing very high throughput, high-quality data, and low process costs. These demands have led to capillary electrophoresis machines with multiple capillaries providing highly parallel analyses, to new electrophoresis matrices, to highly sensitive spectrofluorometers, and to brighter, spectrally distinct fluorescent dyes with which to label DNA. Capillary devices have also been engineered onto microchip formats, on which both the amount of sample required for analysis and the speed of analysis are increased by an order of magnitude. This review examines the advances made in capillary and chip-based microdevices and in the different DNA-based assays developed for mutation detection and genotype analysis using capillary electrophoresis. The automation of attendant processes such as for DNA sample preparation, PCR, and analyte purification are also reviewed. Together, these technological developments provide the throughput demanded by the large genome-sequencing projects.
Asunto(s)
Análisis Mutacional de ADN/instrumentación , Análisis Mutacional de ADN/métodos , Electroforesis Capilar/instrumentación , Electroforesis Capilar/métodos , Polimorfismo Conformacional Retorcido-Simple , Análisis de Secuencia de ADN/instrumentación , Análisis de Secuencia de ADN/métodos , Diseño de Equipo , Microquímica/instrumentación , Microquímica/métodos , Reacción en Cadena de la Polimerasa/instrumentación , Reacción en Cadena de la Polimerasa/métodosRESUMEN
MOTIVATION: A consensus sequence for a family of related sequences is, as the name suggests, a sequence that captures the features common to most members of the family. Consensus sequences are important in various DNA sequencing applications and are a convenient way to characterize a family of molecules. RESULTS: This paper describes a new algorithm for finding a consensus sequence, using the popular optimization method known as simulated annealing. Unlike the conventional approach of finding a consensus sequence by first forming a multiple sequence alignment, this algorithm searches for a sequence that minimises the sum of pairwise distances to each of the input sequences. The resulting consensus sequence can then be used to induce a multiple sequence alignment. The time required by the algorithm scales linearly with the number of input sequences and quadratically with the length of the consensus sequence. We present results demonstrating the high quality of the consensus sequences and alignments produced by the new algorithm. For comparison, we also present similar results obtained using ClustalW. The new algorithm outperforms ClustalW in many cases.