RESUMEN
The Afrotropical mosquito Anopheles gambiae sensu stricto, a major vector of malaria, is currently undergoing speciation into the M and S molecular forms. These forms have diverged in larval ecology and reproductive behavior through unknown genetic mechanisms, despite considerable levels of hybridization. Previous genome-wide scans using gene-based microarrays uncovered divergence between M and S that was largely confined to gene-poor pericentromeric regions, prompting a speciation-with-ongoing-gene-flow model that implicated only about 3% of the genome near centromeres in the speciation process. Here, based on the complete M and S genome sequences, we report widespread and heterogeneous genomic divergence inconsistent with appreciable levels of interform gene flow, suggesting a more advanced speciation process and greater challenges to identify genes critical to initiating that process.
Asunto(s)
Anopheles/genética , Especiación Genética , Genoma de los Insectos , Animales , Anopheles/clasificación , Evolución Molecular , Femenino , Flujo Génico , Masculino , Modelos Genéticos , Polimorfismo de Nucleótido SimpleRESUMEN
The fly Drosophila melanogaster is one of the most intensively studied organisms in biology and serves as a model system for the investigation of many developmental and cellular processes common to higher eukaryotes, including humans. We have determined the nucleotide sequence of nearly all of the approximately 120-megabase euchromatic portion of the Drosophila genome using a whole-genome shotgun sequencing strategy supported by extensive clone-based sequence and a high-quality bacterial artificial chromosome physical map. Efforts are under way to close the remaining gaps; however, the sequence is of sufficient accuracy and contiguity to be declared substantially complete and to support an initial analysis of genome structure and preliminary gene annotation and interpretation. The genome encodes approximately 13,600 genes, somewhat fewer than the smaller Caenorhabditis elegans genome, but with comparable functional diversity.
Asunto(s)
Drosophila melanogaster/genética , Genoma , Análisis de Secuencia de ADN , Animales , Transporte Biológico/genética , Cromatina/genética , Clonación Molecular , Biología Computacional , Mapeo Contig , Sistema Enzimático del Citocromo P-450/genética , Reparación del ADN/genética , Replicación del ADN/genética , Drosophila melanogaster/metabolismo , Eucromatina , Biblioteca de Genes , Genes de Insecto , Heterocromatina/genética , Proteínas de Insectos/química , Proteínas de Insectos/genética , Proteínas de Insectos/fisiología , Proteínas Nucleares/genética , Biosíntesis de Proteínas , Transcripción GenéticaRESUMEN
DNA chip arrays hold considerable promise for diagnostic sequencing of polymerase chain reaction (PCR) products. To date, however, arrays have been relatively expensive, complex to use and difficult to interpret, preventing their adaptation to the clinical lab. A moderate density array method has been developed that enables efficient, easy-to-interpret and robust solid-phase PCR product sequencing. Here, the results of Mycobacterium tuberculosis rifampin resistance mutation detection by primer-extension-based sequence scanning of the rpo B gene of M. tuberculosis are presented. Rifampin resistant clinical isolates were identified in as little as 1 h post PCR amplification with visual results detection.
Asunto(s)
Proteínas Bacterianas/genética , Análisis Mutacional de ADN/métodos , ADN Bacteriano/genética , Farmacorresistencia Microbiana/genética , Mycobacterium tuberculosis/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proteínas de Plantas/genética , Rifampin/farmacología , Tuberculosis/microbiología , Codón/genética , Cartilla de ADN , ARN Polimerasas Dirigidas por ADN , Genes Bacterianos , Genotipo , Humanos , Procesamiento de Imagen Asistido por Computador , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Programas Informáticos , Factores de Tiempo , Tuberculosis/tratamiento farmacológicoRESUMEN
The covalent attachment of disulfide-modified oligonucleotides to a mercaptosilane-modified glass surface is described. This method provides an efficient and specific covalent attachment chemistry for immobilization of DNA probes onto a solid support. Glass slides were derivatized with 3-mercaptopropyl silane for attachment of 5-prime disulfide-modified oligonucleotides via disulfide bonds. An attachment density of approximately 3 x 10(5) oligonucleotides/microm2 was observed. Oligonucleotides attached by this method provided a highly efficient substrate for nucleic acid hybridization and primer extension assays. In addition, we have demonstrated patterning of multiple DNA probes on a glass surface utilizing this attachment chemistry, which allows for array densities of at least 20,000 spots/cm2.
Asunto(s)
Vidrio/química , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sondas de Oligonucleótidos/química , Sondas de ADN , Disulfuros/química , Concentración de Iones de Hidrógeno , Sensibilidad y Especificidad , Compuestos de Sulfhidrilo/química , Factores de TiempoAsunto(s)
Bioensayo/métodos , Biotina/análisis , Animales , Unión Competitiva , Humanos , Sensibilidad y EspecificidadRESUMEN
There is a growing and significant demand for reliable, simple and sensitive methods for repeated scanning of a given gene or gene fragment for detection and characterization of mutations. Solid-phase sequencing by single base primer extension of nested GBATM primers on miniaturized DNA arrays can be used to effectively scan targeted sequences for missense, insertion and deletion mutations. This paper describes the use of N-GBA arrays designed to scan the sequence of a 33 base region of exon 8 of the p53 gene (codons 272-282) encompassing a hot spot for mutations associated with the development of cancer. Synthetic DNA templates containing various missense, insertion and deletion mutations, as well as DNA prepared from pancreatic and biliary tumor cells, were genotyped using the exon 8 arrays.
Asunto(s)
Análisis Mutacional de ADN/métodos , Genes p53 , Adenocarcinoma/química , Animales , Neoplasias del Sistema Biliar/química , Codón/genética , Cartilla de ADN , ADN de Neoplasias/genética , Humanos , Ratones , Mutagénesis Insercional , Trasplante de Neoplasias , Neoplasias Pancreáticas/química , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia , Moldes GenéticosRESUMEN
A reliable, simple, and cost-effective method for the immobilization of relatively short (12-30 mer) oligonucleotide probes to 96-well polystyrene plates was required in our laboratory for use in DNA hybridization-based assays. We compared three different approaches to achieve this immobilization. Two of them are modifications of previously published procedures, requiring the use of modified oligonucleotides and/or modified plates. These were compared to a method developed in our laboratory, whereby passive immobilization occurs by incubation in the presence of salt or a cationic detergent. While all methods resulted in the productive binding of the DNA probes and could therefore be used for hybridization, only the passive immobilization approach met our strict performance criteria and was implemented for use in our DNA genotyping laboratory. It was found that the immobilization in the presence of cationic detergents takes place via a unique mechanism.
Asunto(s)
Cartilla de ADN/química , Hibridación de Ácido Nucleico , Poliestirenos , Secuencia de Bases , Biotina , Cetrimonio , Compuestos de Cetrimonio , Reactivos de Enlaces Cruzados , ADN/biosíntesis , ADN Polimerasa I/química , Detergentes , Dimetilaminas/química , Etildimetilaminopropil Carbodiimida , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Cloruro de SodioRESUMEN
A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.
Asunto(s)
ADN/análisis , Polimorfismo Genético , Autoanálisis , Secuencia de Bases , Biotina , Colorimetría , ADN/química , ADN Polimerasa I/metabolismo , Cartilla de ADN , ADN de Cadena Simple , ADN Polimerasa Dirigida por ADN/metabolismo , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/enzimología , Exodesoxirribonucleasas/metabolismo , Fluoresceína , Fluoresceínas , Colorantes Fluorescentes , Indicadores y Reactivos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Moldes GenéticosRESUMEN
The effect of phosphorothioate bonds on the hydrolytic activity of the 5'-->3' double-strand-specific T7 gene 6 exonuclease was studied. Double-stranded DNA substrates containing one phosphorothioate residue at the 5' end were found to be hydrolyzed by this enzyme as efficiently as unmodified ones. The enzyme activity was, however, completely inhibited by the presence of four phosphorothioates. On the basis of these results, a method for the conversion of double-stranded PCR products into full-length, single-stranded DNA fragments was developed. In this method, one of the PCR primers contains four phosphorothioates at its 5' end, and the opposite strand primer is unmodified. Following the amplification, the double-stranded product is treated with T7 gene 6 exonuclease. The phosphorothioated strand is protected from the action of this enzyme, whereas the opposite strand is hydrolyzed. When the phosphorothioated PCR primer is 5' biotinylated, the single-stranded PCR product can be easily detected colorimetrically after hybridization to an oligonucleotide probe immobilized on a microtiter plate. We also describe a simple and efficient method for the immobilization of relatively short oligonucleotides to microtiter plates with a hydrophilic surface in the presence of salt.