RESUMEN
Ventricular tachycardias (VT) in patients with structural heart diseases have predominantly a scar-associated reentry mechanism so that substrate-based ablation approaches also have to be used in nearly all procedures. In many VT cases-especially in nonischemic cardiomyopathy (NICM) and arrhythmogenic right ventricular cardiomyopathy-a critical epicardial substrate can be identified as an essential component of the reentry circuit so that for the ablation-based modification of the substrate in these cases an epicardial approach is necessary. In cases of redo-VT ablation procedures in ischemic cardiomyopathy (after a previously endocardial ablation), an epicardial approach should also be considered. There are also cases in whom no endocardial substrate can be identified and an isolated epicardial substrate can be identified. Worldwide epicardial VT ablations are usually performed after gaining epicardial access using subxyphoidal puncture. The results of recent studies show a higher efficiency with stabilization of cardiac rhythm and reduction of recurrent VT episodes (about 70% event-free survival at the 2year follow-up) after endo-plus epicardial substrate modification. In electrical storm cases, an early epicardial VT ablation approach also appears to be relevant, especially in NICM. Epicardial instrumentation and ablation represents a complex procedure which should only be performed in experienced centers with cardiac surgery back-up. In these experienced centers, the complications rate is less than 5%.
Asunto(s)
Ablación por Catéter , Pericardio/cirugía , Taquicardia por Reentrada en el Nodo Atrioventricular/cirugía , Cardiomiopatías/diagnóstico , Cardiomiopatías/fisiopatología , Cardiomiopatías/cirugía , Cicatriz/diagnóstico , Cicatriz/fisiopatología , Cicatriz/cirugía , Angiografía Coronaria , Mapeo Epicárdico , Adhesión a Directriz , Humanos , Imagen por Resonancia Magnética , Pericardio/fisiopatología , Reoperación , Taquicardia por Reentrada en el Nodo Atrioventricular/diagnóstico , Taquicardia por Reentrada en el Nodo Atrioventricular/fisiopatología , Resultado del TratamientoRESUMEN
OBJECTIVES: To evaluate the prevalence of injuries of the scapholunate and lunotriquetral interosseous ligaments (SLIL, LTIL) as well as the triangular fibrocartilage complex (TFCC) in intra-articular distal radius fractures (iaDRF). METHODS: Two hundred and thirty-three patients with acute iaDRF underwent MDCT arthrography. The SLIL and LTIL were described as normal, partially or completely ruptured. Major injuries of the SLIL were defined as completely ruptured dorsal segments, those of the LTIL as completely ruptured palmar segments. The TFCC was judged as normal or injured. Interobserver variability was calculated. Injury findings were correlated with the types of iaDRF (AO classification). RESULTS: In 159 patients (68.2 %), no SLIL injuries were seen. Minor SLIL injuries were detected in 54 patients (23.2%), major injuries in 20 patients (8.6%). No correlation was found between the presence of SLIL lesions and the types of iaDRF. Minor LTIL injuries were seen in 23 patients (9.9%), major injuries in only 5 patients (2.2%). The TFCC was altered in 141 patients (60.5%). Interobserver variability was high for MDCT arthrography in assessing SLIL and TFC lesions, and fair for LTIL lesions. CONCLUSION: In iaDRF, prevalence of major injuries of the most relevant SLIL is about 9% as evaluated with CT arthrography. KEY POINTS: The C-shaped SLIL is built of dorsal, middle and palmar segments. In iaDRF, major SLIL injuries are associated in 8.6% of the cases. In iaDRF, the SLIL remains intact in 68.3% of the cases. IaDRF and SLIL ruptures can comprehensively be depicted with MDCT arthrography. A three-compartment approach is recommended to assess intrinsic ligaments and the TFCC.
Asunto(s)
Ligamentos Articulares/diagnóstico por imagen , Ligamentos Articulares/lesiones , Tomografía Computarizada Multidetector , Fracturas del Radio/diagnóstico por imagen , Fibrocartílago Triangular/diagnóstico por imagen , Fibrocartílago Triangular/lesiones , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Artrografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Variaciones Dependientes del Observador , Prevalencia , Radio (Anatomía)/diagnóstico por imagen , Radio (Anatomía)/lesiones , Traumatismos de la Muñeca/diagnóstico por imagen , Articulación de la Muñeca/diagnóstico por imagen , Adulto JovenRESUMEN
PURPOSE: The purpose of this prospective study is to assess the diagnostic value of intravenously applied contrast agent for diagnosing osteonecrosis of the proximal fragment in scaphoid nonunion, and to compare the imaging results with intraoperative findings. MATERIALS AND METHODS: In 88 patients (7 women, 81 men) suffering from symptomatic scaphoid nonunion, preoperative MRI was performed (coronal PD-w FSE fs, sagittal-oblique T1-w SE nonenhanced and T1-w SE fs contrast-enhanced, sagittal T2*-w GRE). MRI interpretation was based on the intensity of contrast enhancement: 0 = none, 1 = focal, 2 = diffuse. Intraoperatively, the osseous viability was scored by means of bleeding points on the osteotomy site of the proximal scaphoid fragment: 0=absent, 1 = moderate, 2 = good. RESULTS: Intraoperatively, 17 necrotic, 29 compromised, and 42 normal proximal fragments were found. In nonenhanced MRI, bone viability was judged necrotic in 1 patient, compromised in 20 patients, and unaffected in 67 patients. Contrast-enhanced MRI revealed 14 necrotic, 21 compromised, and 53 normal proximal fragments. Judging surgical findings as the standard of reference, statistical analysis for nonenhanced MRI was: sensitivity 6.3%, specificity 100%, positive PV 100%, negative PV 82.6%, and accuracy 82.9%; statistics for contrast-enhanced MRI was: sensitivity 76.5%, specificity 98.6%, positive PV 92.9%, negative PV 94.6%, and accuracy 94.3%. Sensitivity for detecting avascular proximal fragments was significantly better (p<0.001) in contrast-enhanced MRI in comparison to nonenhanced MRI. CONCLUSION: Viability of the proximal fragment in scaphoid nonunion can be significantly better assessed with the use of contrast-enhanced MRI as compared to nonenhanced MRI. Bone marrow edema is an inferior indicator of osteonecrosis. Application of intravenous gadolinium is recommended for imaging scaphoid nonunion.
Asunto(s)
Fracturas no Consolidadas/patología , Imagen por Resonancia Magnética/métodos , Compuestos Organometálicos , Osteonecrosis/diagnóstico , Osteonecrosis/cirugía , Hueso Escafoides/lesiones , Hueso Escafoides/patología , Adolescente , Adulto , Medios de Contraste/administración & dosificación , Femenino , Humanos , Aumento de la Imagen/métodos , Inyecciones Intravenosas , Masculino , Persona de Mediana Edad , Compuestos Organometálicos/administración & dosificación , Osteonecrosis/etiología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenRESUMEN
Fractures of the distal radius, which currently are treated with palmar locking plates, are often accompanied by carpal lesions. Tears of the scapholunate interosseus ligament (SL) can affect the outcome. Between January 2007 and May 2008, 28 patients with distal intraarticular fractures of the radius were included in a prospective study. Preoperative CT-arthrography was performed. SL tears were found in 11 patients, with 10 partial and one complete rupture observed. A tear of the triangular fibrocartilage complex (TFCC) was detected in 16 patients. Every patient was operated with a palmar locking plate through a palmar approach between the flexor carpi radialis tendon and the radial artery. Then, a palmar wrist arthroscopy using a palmar portal was performed. Eleven SL tears with 9 partial and two total ruptures were diagnosed by arthroscopy. Ten lesions were associated with a C1-fracture with a fracture line projected onto the scapholunate interval. The TFCC was appraisable by palmar wrist arthroscopy only in 4 patients. Three of the SL tears detected by CT-arthrography could not be confirmed by palmar wrist arthroscopy. One complete rupture and one partial lesion confirmed by palmar wrist arthroscopy were found by CT-arthrography to be intact. Palmar wrist arthroscopy affords certainty when assessing the SL ligament. In this study, an assessment of ulnocarpal structures was not possible. For assessment of the ulnocarpal structures, CT-arthrography was superior to palmar wrist arthroscopy. However, the latter is an alternative during emergency treatment or when CT-arthrography is not available.
Asunto(s)
Artroscopía/métodos , Ligamentos Articulares/lesiones , Hueso Semilunar/lesiones , Fracturas del Radio/cirugía , Hueso Escafoides/lesiones , Fibrocartílago Triangular/cirugía , Traumatismos de la Muñeca/cirugía , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Artrografía , Placas Óseas , Femenino , Fijación Interna de Fracturas , Humanos , Procesamiento de Imagen Asistido por Computador , Ligamentos Articulares/diagnóstico por imagen , Ligamentos Articulares/cirugía , Hueso Semilunar/diagnóstico por imagen , Hueso Semilunar/cirugía , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Fracturas del Radio/diagnóstico por imagen , Rotura , Hueso Escafoides/diagnóstico por imagen , Hueso Escafoides/cirugía , Sensibilidad y Especificidad , Tomografía Computarizada por Rayos X , Fibrocartílago Triangular/diagnóstico por imagen , Traumatismos de la Muñeca/diagnóstico por imagen , Adulto JovenRESUMEN
The partial tear of the scapholunate ligament (pre-dynamic stage of SLD) as well as the complete tear (dynamic stage) does not lead to carpal malalignment. However, if the completely ruptured ligament is accompanied by lesions of the extrinsic ligaments, both the scaphoid and the lunate are malaligned already at rest (static stage of SLD). Later, osteoarthritis will develop, beginning in the radioscaphoid compartment, progressing to the midcarpal joint, and ending in a carpal collapse (osteoarthrotic stage of SLD). Dynamic SLD is detectable only in stress views and in cinematography. The high utility of MRI for directly visualizing the injured ligament is emphasized: reparation tissue is focally enhanced at the rupture site by intravenously applied contrast agent; the individual segments of the scapholunate ligament can be visualized in direct MR arthrography, therefore allowing differentiation of partial and complete ligamentous tears.
Asunto(s)
Inestabilidad de la Articulación/diagnóstico , Ligamentos Articulares/lesiones , Imagen por Resonancia Magnética/métodos , Tomografía Computarizada por Rayos X/métodos , Traumatismos de la Muñeca/diagnóstico , Humanos , Inestabilidad de la Articulación/etiología , Ligamentos Articulares/diagnóstico por imagen , Ligamentos Articulares/patología , Guías de Práctica Clínica como Asunto , Pautas de la Práctica en Medicina , Pronóstico , Rotura/diagnóstico , Traumatismos de la Muñeca/complicacionesRESUMEN
Global patterns of human DNA sequence variation (haplotypes) defined by common single nucleotide polymorphisms (SNPs) have important implications for identifying disease associations and human traits. We have used high-density oligonucleotide arrays, in combination with somatic cell genetics, to identify a large fraction of all common human chromosome 21 SNPs and to directly observe the haplotype structure defined by these SNPs. This structure reveals blocks of limited haplotype diversity in which more than 80% of a global human sample can typically be characterized by only three common haplotypes.
Asunto(s)
Cromosomas Humanos Par 21/genética , Haplotipos/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimorfismo de Nucleótido Simple/genética , Algoritmos , Alelos , Animales , Etnicidad/genética , Frecuencia de los Genes/genética , Variación Genética/genética , Genoma Humano , Humanos , Células Híbridas/metabolismo , Mutación/genética , Grupos Raciales/genética , Distribución Aleatoria , Sensibilidad y EspecificidadRESUMEN
Comparison of human sequences with the DNA of other mammals is an excellent means of identifying functional elements in the human genome. Here we describe the utility of high-density oligonucleotide arrays as a rapid approach for comparing human sequences with the DNA of multiple species whose sequences are not presently available. High-density arrays representing approximately 22.5 Mb of nonrepetitive human chromosome 21 sequence were synthesized and then hybridized with mouse and dog DNA to identify sequences conserved between humans and mice (human-mouse elements) and between humans and dogs (human-dog elements). Our data show that sequence comparison of multiple species provides a powerful empiric method for identifying actively conserved elements in the human genome. A large fraction of these evolutionarily conserved elements are present in regions on chromosome 21 that do not encode known genes.
Asunto(s)
Cromosomas Humanos Par 21/genética , Secuencia Conservada/genética , Evolución Molecular , Animales , Cromosomas Artificiales Bacterianos/genética , ADN/genética , Perros , Genes Sobrepuestos/genética , Humanos , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Sensibilidad y Especificidad , Sintenía/genéticaRESUMEN
Atomic force microscopy (AFM) can be used to probe the mechanics of molecular recognition between surfaces. In the application known as "chemical force" microscopy (CFM), a chemically modified AFM tip probes a surface through chemical recognition. When modified with a biological ligand or receptor, the AFM tip can discriminate between its biological binding partner and other molecules on a heterogeneous substrate. The strength of the interaction between the modified tip and the substrate is governed by the molecular affinity. We have used CFM to probe the interactions between short segments of single-strand DNA (oligonucleotides). First, a latex microparticle was modified with the sequence 3'-CAGTTCTACGATGGCAAGTC and epoxied to a standard AFM cantilever. This DNA-modified probe was then used to scan substrates containing the complementary sequence 5'-GTCAAGATGCTACCGTTCAG. These substrates consisted of micron-scale, patterned arrays of one or more distinct oligonucleotides. A strong friction interaction was measured between the modified tip and both elements of surface-bound DNA. Complementary oligonucleotides exhibited a stronger friction than the noncomplementary sequences within the patterned array. The friction force correlated with the measured strength of adhesion (rupture force) for the tip- and array-bound oligonucleotides. This result is consistent with the formation of a greater number of hydrogen bonds for the complementary sequence, suggesting that the friction arises from a sequence-specific interaction (hybridization) of the tip and surface DNA.
Asunto(s)
ADN/química , Microscopía de Fuerza Atómica/métodos , Hibridación de Ácido Nucleico , Secuencia de Bases , Fenómenos Biofísicos , Biofisica , ADN/ultraestructura , Sondas de ADN/química , Sondas de ADN/genética , Látex , Microscopía de Fuerza Atómica/instrumentación , MicroesferasRESUMEN
Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.
Asunto(s)
Hominidae/genética , Polimorfismo Genético , Alelos , Animales , Fosfatos de Dinucleósidos/química , Fosfatos de Dinucleósidos/genética , Genotipo , Gorilla gorilla/genética , Humanos , Modelos Genéticos , Pan troglodytes/genética , LinajeRESUMEN
Experimental genomics involves taking advantage of sequence information to investigate and understand the workings of genes, cells and organisms. We have developed an approach in which sequence information is used directly to design high-density, two-dimensional rays of synthetic oligonucleotides. The GeneChipe probe arrays are made using spatially patterned, light-directed combinatorial chemical synthesis and contain up to hundreds of thousands of different oligonucleotides on a small glass surface. The arrays have been designed and used for quantitative and highly parallel measurements of gene expression, to discover polymorphic loci and to detect the presence of thousands of alternative alleles. Here, we describe the fabrication of the arrays, their design and some specific applications to high-throughput genetic and cellular analysis.
Asunto(s)
Expresión Génica , Genotipo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligonucleótidos/síntesis química , Animales , Secuencia de Bases , Sistemas de Administración de Bases de Datos , HumanosRESUMEN
Pairs of high density oligonucleotide arrays (DNA chips) consisting of >96 000 oligonucleotides were designed to screen the entire 5.53 kb coding region of the hereditary breast and ovarian cancer BRCA1 gene for all possible sequence changes in the homozygous and heterozygous states. Single-stranded RNA targets were generated by PCR amplification of individual BRCA1 exons using primers containing T3 and T7RNA polymerase promoter tails followed by in vitro transcription and partial fragmentation reactions. Fluorescent hybridization signals from targets containing the four natural bases to >5592 different fully complementary 25mer oligonucleotide probes on the chip varied over two orders of magnitude. To examine the thermodynamic contribution of rU.dA and rA.dT target.probe base pairs to this variability, modified uridine [5-methyluridine and 5-(1-propynyl)-uridine)] and modified adenosine (2,6-diaminopurine riboside) 5'-triphosphates were incorporated into BRCA1 targets. Hybridization specificity was assessed based upon hybridization signals from >33 200 probes containing centrally localized single base pair mismatches relative to target sequence. Targets containing 5-methyluridine displayed promising localized enhancements in hybridization signal, especially in pyrimidine-rich target tracts, while maintaining single nucleotide mismatch hybridization specificities comparable with those of unmodified targets.
Asunto(s)
Genes BRCA1 , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Disparidad de Par Base , Secuencia de Bases , Neoplasias de la Mama/genética , Cartilla de ADN/genética , ADN de Neoplasias/genética , Femenino , Heterocigoto , Homocigoto , Humanos , Técnicas In Vitro , Hibridación de Ácido Nucleico , Nucleótidos , Sondas de Oligonucleótidos/genética , Neoplasias Ováricas/genética , ARN Neoplásico/genética , Transcripción GenéticaRESUMEN
High density oligonucleotide arrays (DNA chips) have been used in two color mutational analysis of the 3.43 kb exon 11 of the hereditary breast and ovarian cancer gene BRCA1 . Two color analysis allows competitive hybridization between a reference standard and an unknown sample, improving the performance of the assay. Fluorescein and phycoerythrin dyes werepreviously used due to their compatibility with a single line 488 nm excitation source. Here we show that an alternative dye combination, containing the energy transfer dye system phycoerythrin*cy5 along with phycoerythrin, provides more evenly matched signal intensities and decreased spectral overlap between the two fluorophores, while maintaining compatibility with a 488 nm excitation source.
Asunto(s)
Colorantes , Análisis Mutacional de ADN/métodos , Genes BRCA1 , Hibridación de Ácido Nucleico/métodos , Oligodesoxirribonucleótidos/genética , Secuencia de Bases , Exones , Femenino , Colorantes Fluorescentes , Heterocigoto , Humanos , Ficoeritrina , Polimorfismo GenéticoRESUMEN
We explored the utility of high-density oligonucleotide arrays (DNA chips) for obtaining sequence information from homologous genes in closely related species. Orthologues of the human BRCA1 exon 11, all approximately 3.4 kb in length and ranging from 98.2% to 83.5% nucleotide identity, were subjected to hybridization-based and conventional dideoxysequencing analysis. Retrospective guidelines for identifying high-fidelity hybridization-based sequence calls were formulated based upon dideoxysequencing results. Prospective application of these rules yielded base-calling with at least 98.8% accuracy over orthologous sequence tracts shown to have approximately 99% identity. For higher primate sequences with greater than 97% nucleotide identity, base-calling was made with at least 99.91% accuracy covering a minimum of 97% of the sequence. Using a second-tier confirmatory hybridization chip strategy, shown in several cases to confirm the identity of predicted sequence changes, the complete sequence of the chimpanzee, gorilla and orangutan orthologues should be deducible solely through hybridization-based methodologies. Analysis of less highly conserved orthologues can still identify conserved nucleotide tracts of at least 15 nucleotides and can provide useful information for designing primers. DNA-chip based assays can be a valuable new technology for obtaining high-throughput cost-effective sequence information from related genomes.
Asunto(s)
Proteína BRCA1/genética , Evolución Molecular , Genes BRCA1 , Primates/genética , Alouatta , Animales , Secuencia de Bases , Cartilla de ADN , Perros , Exones , Galago , Técnicas Genéticas , Gorilla gorilla , Hominidae , Humanos , Lemur , Macaca mulatta , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Pongo pygmaeus , Primates/clasificaciónRESUMEN
Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.
Asunto(s)
Análisis Mutacional de ADN/métodos , Leucina Zippers/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Fosfatidilinositol 3-Quinasas/genética , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Exones/genética , Femenino , Proyecto Genoma Humano , Humanos , Masculino , Mutación/genética , Sensibilidad y Especificidad , Proteínas Supresoras de TumorAsunto(s)
Esocidae/virología , Enfermedades de los Peces , Filogenia , Retroviridae/clasificación , Retroviridae/genética , Sarcoma/veterinaria , Neoplasias Cutáneas/veterinaria , Secuencia de Aminoácidos , Animales , Productos del Gen env/biosíntesis , Productos del Gen env/genética , Genes gag , Datos de Secuencia Molecular , ADN Polimerasa Dirigida por ARN/química , ADN Polimerasa Dirigida por ARN/genética , Retroviridae/aislamiento & purificación , Sarcoma/virología , Homología de Secuencia de Aminoácido , Neoplasias Cutáneas/virología , Transcripción GenéticaRESUMEN
The ability to scan a large gene rapidly and accurately for all possible heterozygous mutations in large numbers of patient samples will be critical for the future of medicine. We have designed high-density arrays consisting of over 96,600 oligonucleotides 20-nucleotides (nt) in length to screen for a wide range of heterozygous mutations in the 3.45-kilobases (kb) exon 11 of the hereditary breast and ovarian cancer gene BRCA1. Reference and test samples were co-hybridized to these arrays and differences in hybridization patterns quantitated by two-colour analysis. Fourteen of fifteen patient samples with known mutations were accurately diagnosed, and no false positive mutations were identified in 20 control samples. Eight single nucleotide polymorphisms were also readily detected. DNA chip-based assays may provide a valuable new technology for high-throughput cost-efficient detection of genetic alterations.
Asunto(s)
Proteína BRCA1/genética , Sondas de ADN , Heterocigoto , Mutación , Reacciones Falso Negativas , Estudios de Factibilidad , Fluoresceína , Fluoresceínas , Fluorescencia , Tamización de Portadores Genéticos , Humanos , Hibridación de Ácido Nucleico , Sensibilidad y EspecificidadRESUMEN
Rapid access to genetic information is central to the revolution taking place in molecular genetics. The simultaneous analysis of the entire human mitochondrial genome is described here. DNA arrays containing up to 135,000 probes complementary to the 16.6-kilobase human mitochondrial genome were generated by light-directed chemical synthesis. A two-color labeling scheme was developed that allows simultaneous comparison of a polymorphic target to a reference DNA or RNA. Complete hybridization patterns were revealed in a matter of minutes. Sequence polymorphisms were detected with single-base resolution and unprecedented efficiency. The methods described are generic and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability.
Asunto(s)
ADN Mitocondrial/genética , Genoma , Mitocondrias/genética , Hibridación de Ácido Nucleico , Sondas de Oligonucleótidos , Algoritmos , Composición de Base , Secuencia de Bases , Clonación Molecular , Fluoresceína , Fluoresceínas , Expresión Génica , Variación Genética , Humanos , Ficoeritrina , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Análisis de Secuencia de ADNRESUMEN
As the Human Genome Project and related efforts identify and determine the DNA sequences of human genes, it is important that highly reliable and efficient mechanisms are found to access individual genetic variation. It is only through a greater understanding of genetic diversity that the true benefit of the Human Genome Project will be realized. One approach, hybridization to high-density arrays of oligonucleotides, is a fast and effective means of accessing this genetic variation. Light-directed chemical synthesis has been used to generate miniaturized, high-density arrays of oligonucleotide probes. Application-specific oligonucleotide probe array designs have been developed for the rapid screening of characterized genes. Dedicated instrumentation and software have been developed for array hybridization, fluorescence detection and data acquisition and analysis. In a specific and challenging application, oligonucleotide probe arrays have been used to screen the reverse transcriptase and protease genes of the highly polymorphic HIV-1 genome to explore genetic diversity and detect mutations conferring resistance to antiviral drugs. Results from this application strongly suggest that oligonucleotide probe arrays will be a powerful tool for rapid investigations in sequence checking, pathogen detection, expression monitoring and DNA molecular recognition.
Asunto(s)
Variación Genética , Sondas de Oligonucleótidos , Secuencia de Bases , Colorantes Fluorescentes , VIH-1/genética , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Polimorfismo GenéticoRESUMEN
Walleye dermal sarcoma virus (WDSV) is a fish retrovirus associated with the development of tumors in walleyes. We have determined the complete nucleotide sequence of a DNA clone of WDSV, the N-terminal amino acid sequences of the major proteins, and the start site for transcription. The long terminal repeat is 590 bp in length, with the U3 region containing consensus sequences likely to be involved in viral gene expression. A predicted histidyl-tRNA binding site is located 3 nucleotides distal to the 3' end of the long terminal repeat. Virus particles purified by isopycnic sedimentation followed by rate zonal sedimentation showed major polypeptides with molecular sizes of 90, 25, 20, 14, and 10 kDa. N-terminal sequencing of these allowed unambiguous assignment of the small polypeptides as products of the gag gene, including CA and NC, and the large polypeptide as the TM product of env. The 582-amino-acid (aa) Gag protein precursor is predicted to be myristylated as is found for most retroviruses. NC contains a single Cys-His motif like those found in all retroviruses except spumaviruses. The WDSV pro and pol genes are in the same translational reading frame as gag and thus apparently are translated after termination suppression. The env gene encodes a surface (SU) protein of 469 aa predicted to be highly glycosylated and a large transmembrane (TM) protein of 754 aa. The sequence of TM is unusual in that it ends in a very hydrophobic segment of 65 residues containing a single charged residue. Following the env gene are two nonoverlapping long open reading frames of 290 aa (orf-A) and 306 aa (orf-B), neither of which shows significant sequence similarity with known genes. A third open reading frame of 119 aa (orf-C) is located in the leader region preceding gag. The predicted amino acid sequence of reverse transcriptase would place WDSV phylogenetically closest to the murine leukemia virus-related genus of retroviruses. However, other members of this genus do not have accessory genes, suggesting that WDSV acquired orf-A, orf-B, and perhaps orf-C late in its evolution. We hypothesize by analogy with other complex retroviruses that the accessory genes of WDSV function in the regulation of transcription and in RNA processing and also in the induction of walleye dermal sarcoma.
Asunto(s)
ADN Viral/genética , Enfermedades de los Peces , Genes Virales , Retroviridae/genética , Sarcoma/veterinaria , Neoplasias Cutáneas/veterinaria , Proteínas Virales/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cartilla de ADN , ADN Viral/química , Elementos de Facilitación Genéticos , Peces , Genes gag , Genes pol , Datos de Secuencia Molecular , Filogenia , Sistemas de Lectura , Retroviridae/química , Sarcoma/virología , Neoplasias Cutáneas/virología , Transcripción Genética , Proteínas Virales/químicaRESUMEN
We describe here a method for constructing ordered molecular arrays and for detecting binding of biomolecules to these arrays using atomic force microscopy (AFM). These arrays simplify the discrimination of surface-bound biomolecules through the spatial control of ligand presentation. First, photolithography is used to spatially direct the synthesis of a matrix of biological ligands. A high-affinity binding partner is then applied to the matrix, which binds at locations defined by the ligand array. AFM is then used to detect the presence and organization of the high-affinity binding partner. Streptavidin-biotin arrays of 100 x 100 microns and 8 x 8 microns elements were fabricated by this method. Contact and noncontact AFM images reveal a dense lawn of streptavidin specific to the regions of biotin derivatization. These protein regions are characterized by a height profile of approximately 40 A over the base substrate with a 350-nm edge corresponding to the diffraction zone of the photolithography. High resolution scans reveal a granular topography dominated by 300 A diameter features. The ligand-bound protein can then be etched from the substrate using the AFM tip, leaving an 8 A shelf that probably corresponds to the underlying biotin layer.