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1.
Proc Natl Acad Sci U S A ; 106(43): 18125-30, 2009 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-19815517

RESUMO

DNA hybridization plays a central role in biology and, increasingly, in materials science. Yet, there is no precedent for examining the pathways by which specific single-stranded DNA sequences interact to assemble into a double helix. A detailed model of DNA is adopted in this work to examine such pathways and to determine the role of sequence, if any, on DNA hybridization. Transition path sampling simulations reveal that DNA rehybridization is prompted by a distinct nucleation event involving molecular sites with approximately four bases pairing with partners slightly offset from those involved in ideal duplexation. Nucleation is promoted in regions with repetitive base pair sequence motifs, which yield multiple possibilities for finding complementary base partners. Repetitive sequences follow a nonspecific pathway to renaturation consistent with a molecular "slithering" mechanism, whereas random sequences favor a restrictive pathway involving the formation of key base pairs before renaturation fully ensues.


Assuntos
DNA/química , Oligonucleotídeos/química , Transição de Fase , Pareamento de Bases , Sequência de Bases , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Temperatura de Transição
2.
Biophys J ; 96(5): 1675-90, 2009 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-19254530

RESUMO

A mesoscale model of DNA is presented (3SPN.1), extending the scheme previously developed by our group. Each nucleotide is mapped onto three interaction sites. Solvent is accounted for implicitly through a medium-effective dielectric constant and electrostatic interactions are treated at the level of Debye-Hückel theory. The force field includes a weak, solvent-induced attraction, which helps mediate the renaturation of DNA. Model parameterization is accomplished through replica exchange molecular dynamics simulations of short oligonucleotide sequences over a range of composition and chain length. The model describes the melting temperature of DNA as a function of composition as well as ionic strength, and is consistent with heat capacity profiles from experiments. The dependence of persistence length on ionic strength is also captured by the force field. The proposed model is used to examine the renaturation of DNA. It is found that a typical renaturation event occurs through a nucleation step, whereby an interplay between repulsive electrostatic interactions and colloidal-like attractions allows the system to undergo a series of rearrangements before complete molecular reassociation occurs.


Assuntos
DNA/química , Modelos Químicos , Renaturação de Ácido Nucleico , Algoritmos , Simulação por Computador , Temperatura Alta , Modelos Moleculares , Concentração Osmolar , Eletricidade Estática , Temperatura de Transição
3.
Macromolecules ; 38(15): 6680-6687, 2005.
Artigo em Inglês | MEDLINE | ID: mdl-19057656

RESUMO

The conformation and dynamics of a single DNA molecule undergoing oscillatory pressure-driven flow in microfluidic channels is studied using Brownian dynamics simulations, accounting for hydrodynamic interactions between segments in the bulk and between the chain and the walls. Oscillatory flow provides a scenario under which the polymers may remain in the channel for an indefinite amount of time as they are stretched and migrate away from the channel walls. We show that by controlling the chain length, flow rate and oscillatory flow frequency, we are able to manipulate the chain extension and the chain migration from the channel walls. The chain stretch and the chain depletion layer thickness near the wall are found to increase as the Weissenberg number increases and as the oscillatory frequency decreases.

4.
Infect Immun ; 71(5): 2775-86, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12704152

RESUMO

We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.


Assuntos
Genoma Bacteriano , Genômica , Shigella flexneri/genética , Sequência de Bases , Elementos de DNA Transponíveis , Genes Bacterianos , Dados de Sequência Molecular , Filogenia , Plasmídeos , Shigella flexneri/classificação , Shigella flexneri/patogenicidade
5.
Proc Natl Acad Sci U S A ; 99(26): 17020-4, 2002 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-12471157

RESUMO

We present the complete genome sequence of uropathogenic Escherichia coli, strain CFT073. A three-way genome comparison of the CFT073, enterohemorrhagic E. coli EDL933, and laboratory strain MG1655 reveals that, amazingly, only 39.2% of their combined (nonredundant) set of proteins actually are common to all three strains. The pathogen genomes are as different from each other as each pathogen is from the benign strain. The difference in disease potential between O157:H7 and CFT073 is reflected in the absence of genes for type III secretion system or phage- and plasmid-encoded toxins found in some classes of diarrheagenic E. coli. The CFT073 genome is particularly rich in genes that encode potential fimbrial adhesins, autotransporters, iron-sequestration systems, and phase-switch recombinases. Striking differences exist between the large pathogenicity islands of CFT073 and two other well-studied uropathogenic E. coli strains, J96 and 536. Comparisons indicate that extraintestinal pathogenic E. coli arose independently from multiple clonal lineages. The different E. coli pathotypes have maintained a remarkable synteny of common, vertically evolved genes, whereas many islands interrupting this common backbone have been acquired by different horizontal transfer events in each strain.


Assuntos
Escherichia coli/genética , Genoma Bacteriano , Pielonefrite/microbiologia , Doença Aguda , Sequência de Bases , Escherichia coli/patogenicidade , Feminino , Estruturas Genéticas , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta
6.
Genome Res ; 11(9): 1584-93, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11544203

RESUMO

We have constructed NheI and XhoI optical maps of Escherichia coli O157:H7 solely from genomic DNA molecules to provide a uniquely valuable scaffold for contig closure and sequence validation. E. coli O157:H7 is a common pathogen found in contaminated food and water. Our approach obviated the need for the analysis of clones, PCR products, and hybridizations, because maps were constructed from ensembles of single DNA molecules. Shotgun sequencing of bacterial genomes remains labor-intensive, despite advances in sequencing technology. This is partly due to manual intervention required during the last stages of finishing. The applicability of optical mapping to this problem was enhanced by advances in machine vision techniques that improved mapping throughput and created a path to full automation of mapping. Comparisons were made between maps and sequence data that characterized sequence gaps and guided nascent assemblies.


Assuntos
Mapeamento de Sequências Contíguas/métodos , Escherichia coli O157/genética , Genoma Bacteriano , Mapeamento por Restrição/métodos , Análise de Sequência de DNA/métodos , Software
7.
Am J Med Sci ; 321(5): 352-4, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11370800

RESUMO

Papillary cystic and solid tumor of the pancreas is a rare neoplasm with low malignancy potential generally found in young women. Although its presentation is typically one of vague abdominal complaints, its radiographic and histologic characteristics are distinct. Recognition of the clinical and pathological spectrum of papillary cystic and solid tumor of the pancreas is essential for diagnosing this uncommon condition and differentiating it from other pancreatic masses encountered in the young.


Assuntos
Dor Abdominal/etiologia , Cistadenoma Papilar/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Adulto , Cistadenoma Papilar/complicações , Cistadenoma Papilar/cirurgia , Feminino , Humanos , Neoplasias Pancreáticas/complicações , Neoplasias Pancreáticas/cirurgia
8.
Nature ; 409(6819): 529-33, 2001 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-11206551

RESUMO

The bacterium Escherichia coli O157:H7 is a worldwide threat to public health and has been implicated in many outbreaks of haemorrhagic colitis, some of which included fatalities caused by haemolytic uraemic syndrome. Close to 75,000 cases of O157:H7 infection are now estimated to occur annually in the United States. The severity of disease, the lack of effective treatment and the potential for large-scale outbreaks from contaminated food supplies have propelled intensive research on the pathogenesis and detection of E. coli O157:H7 (ref. 4). Here we have sequenced the genome of E. coli O157:H7 to identify candidate genes responsible for pathogenesis, to develop better methods of strain detection and to advance our understanding of the evolution of E. coli, through comparison with the genome of the non-pathogenic laboratory strain E. coli K-12 (ref. 5). We find that lateral gene transfer is far more extensive than previously anticipated. In fact, 1,387 new genes encoded in strain-specific clusters of diverse sizes were found in O157:H7. These include candidate virulence factors, alternative metabolic capacities, several prophages and other new functions--all of which could be targets for surveillance.


Assuntos
Escherichia coli O157/genética , Genoma Bacteriano , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/patogenicidade , Variação Genética , Humanos , Dados de Sequência Molecular , Polimorfismo Genético , Análise de Sequência de DNA , Especificidade da Espécie , Virulência/genética
9.
Mol Cell Biol ; 20(21): 7933-42, 2000 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11027264

RESUMO

A major pathway of eukaryotic mRNA turnover occurs by deadenylation-dependent decapping that exposes the transcript to 5'-->3' exonucleolytic degradation. A critical step in this pathway is decapping, since removal of the cap structure permits 5'-->3' exonucleolytic digestion. Based on alterations in mRNA decay rate from strains deficient in translation initiation, it has been proposed that the decapping rate is modulated by a competition between the cytoplasmic cap binding complex, which promotes translation initiation, and the decapping enzyme, Dcp1p. In order to test this model directly, we examined the functional interaction of Dcp1p and the cap binding protein, eukaryotic translation initiation factor 4E (eIF4E), in vitro. These experiments indicated that eIF4E is an inhibitor of Dcp1p in vitro due to its ability to bind the 5' cap structure. In addition, we demonstrate that in vivo a temperature-sensitive allele of eIF4E (cdc33-42) suppressed the decapping defect of a partial loss-of-function allele of DCP1. These results argue that dissociation of eIF4E from the cap structure is required before decapping. Interestingly, the temperature-sensitive allele of eIF4E does not suppress the decapping defect seen in strains lacking the decapping activators, Lsm1p and Pat1p. This indicates that these activators of decapping affect a step in mRNA turnover distinct from the competition between Dcp1 and eIF4E.


Assuntos
Endorribonucleases , Proteínas Fúngicas/metabolismo , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae , Alelos , Northern Blotting , Fator de Iniciação 4E em Eucariotos , Proteínas Fúngicas/genética , Vetores Genéticos , Genótipo , Glutationa Transferase/metabolismo , Modelos Genéticos , Mutação , Ligação Proteica , Biossíntese de Proteínas , Proteínas de Ligação ao Cap de RNA , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Temperatura , Fatores de Tempo , Transcrição Gênica
10.
Genome Res ; 10(9): 1421-9, 2000 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-10984460

RESUMO

The accurate mapping of clones derived from genomic regions containing complex arrangements of repeated elements presents special problems for DNA sequencers. Recent advances in the automation of optical mapping have enabled us to map a set of 16 BAC clones derived from the DAZ locus of the human Y chromosome long arm, a locus in which the entire DAZ gene as well as subsections within the gene copies have been duplicated. High-resolution optical mapping employing seven enzymes places these clones into two contigs representing four distinct copies of the DAZ gene and highlights a number of differences between individual copies of DAZ.


Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Bacterianos/genética , Proteínas de Ligação a RNA/genética , Cromossomo Y/genética , Mapeamento Cromossômico/instrumentação , Clonagem Molecular/métodos , Mapeamento de Sequências Contíguas , Proteína 1 Suprimida em Azoospermia , Desoxirribonucleases de Sítio Específico do Tipo II/análise , Marcadores Genéticos/genética , Humanos , Mutagênese Insercional , Proteínas de Ligação a RNA/análise , Mapeamento por Restrição
11.
Int J Biol Markers ; 15(4): 324-9, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11192828

RESUMO

We have evaluated the performance of the Bayer Immuno 1 serum HER-2/neu assay. The precision is excellent and varied between 1.7% and 2.1% at values of HER-2/neu ranging from 16.8 ng/mL to 108.6 ng/mL. In normal women who were followed on a monthly basis the average deviation was 6%. The concentration (mean +/- SD) in normal women was 8.7 +/- 3.2 ng/mL. There was no difference between pre and postmenopausal women. The normal/abnormal cutoff was defined as greater than 15 ng/mL. In normal women and those with benign disease the specificity varied between 95% and 100%. In women with breast cancer the sensitivity was 1.7% in stage I disease, 3.0% in stage II, 16.7 in stage III and 35.5% in stage IV. There were 56 elevations (14.7%) in the total group of 285 women with breast cancer. There was an excellent correlation with a microtitre plate method (r=0.9944). Longitudinal studies showed clearly that women who expressed HER-2/neu in their tissue had elevated serum levels and these levels reflected the clinical course of the patient. More extensive control studies are required to establish the role of HER-2/neu assays in the management of women with breast cancer.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/sangue , Neoplasias da Mama/tratamento farmacológico , Receptor ErbB-2/sangue , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/sangue , Antígeno Carcinoembrionário/análise , Monitoramento de Medicamentos , Feminino , Humanos , Estudos Longitudinais , Mucina-1/análise , Metástase Neoplásica , Estadiamento de Neoplasias , Kit de Reagentes para Diagnóstico , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Trastuzumab
12.
Nat Genet ; 23(3): 309-13, 1999 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-10610179

RESUMO

The unicellular parasite Plasmodium falciparum is the cause of human malaria, resulting in 1.7-2.5 million deaths each year. To develop new means to treat or prevent malaria, the Malaria Genome Consortium was formed to sequence and annotate the entire 24.6-Mb genome. The plan, already underway, is to sequence libraries created from chromosomal DNA separated by pulsed-field gel electrophoresis (PFGE). The AT-rich genome of P. falciparum presents problems in terms of reliable library construction and the relative paucity of dense physical markers or extensive genetic resources. To deal with these problems, we reasoned that a high-resolution, ordered restriction map covering the entire genome could serve as a scaffold for the alignment and verification of sequence contigs developed by members of the consortium. Thus optical mapping was advanced to use simply extracted, unfractionated genomic DNA as its principal substrate. Ordered restriction maps (BamHI and NheI) derived from single molecules were assembled into 14 deep contigs corresponding to the molecular karyotype determined by PFGE (ref. 3).


Assuntos
Genoma de Protozoário , Mapeamento Físico do Cromossomo/métodos , Plasmodium falciparum/genética , Animais , Cromossomos/genética , Cromossomos Artificiais de Levedura/genética , Mapeamento de Sequências Contíguas/métodos , Eletroforese em Gel de Campo Pulsado , Etiquetas de Sequências Expressas , Biblioteca Genômica , Processamento de Imagem Assistida por Computador , Cariotipagem/métodos , Óptica e Fotônica , Reprodutibilidade dos Testes , Mapeamento por Restrição/métodos , Sensibilidade e Especificidade
13.
Mamm Genome ; 10(10): 1005-9, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10501971

RESUMO

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors.


Assuntos
Óptica e Fotônica , Reação em Cadeia da Polimerase/métodos , Mapeamento por Restrição/métodos , Enzimas de Restrição do DNA/metabolismo , Genoma Humano , Humanos , Processamento de Imagem Assistida por Computador , Mapeamento Físico do Cromossomo/métodos , Ativador de Plasminogênio Tecidual/genética
14.
Science ; 285(5433): 1558-62, 1999 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-10477518

RESUMO

A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis. Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps. A third smaller chromosome (176 kilobases) was identified and characterized. Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after gamma irradiation.


Assuntos
Mapeamento de Sequências Contíguas/métodos , Genoma Bacteriano , Cocos Gram-Positivos/genética , Mapeamento por Restrição/métodos , Cromossomos Bacterianos , Dano ao DNA , Reparo do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Bacteriano/efeitos da radiação , DNA Circular/química , Raios gama , Cocos Gram-Positivos/efeitos da radiação , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Conformação de Ácido Nucleico , Plasmídeos , Tolerância a Radiação , Recombinação Genética
15.
Mol Cell Biol ; 19(8): 5247-56, 1999 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-10409716

RESUMO

The turnover of most mRNAs in Saccharomyces cerevisiae begins with deadenylation followed by decapping and 5'-->3' exonucleolytic digestion. An important question involves the mechanisms that allow particular mRNAs to exhibit different rates of both deadenylation and decapping. Since the cap structure plays a critical role in the assembly of translation initiation factors, we hypothesized that the status of the cytoplasmic cap binding complex would affect the rate of decapping. To test this hypothesis, we examined mRNA decay rates in yeast strains that were defective in several translation initiation factors that are part of the cap binding complex. These experiments yielded three significant observations. First, any mutation known to inhibit translation initiation also increased the rate of decapping. Second, decapping still occurred only after deadenylation, suggesting that the ability of the poly(A) tail to inhibit decapping does not require efficient translation of the transcript. Third, mutants with defects in translation initiation factors also showed an increase in the rate of deadenylation, suggesting that the rate of deadenylation may be controlled primarily by the translation status of the transcript. These results argue that the nature of the translation initiation complex is a critical factor in determining the mRNA half-life. This view also implies that some cis-acting sequences that modulate mRNA decay rate do so by affecting the translation status of the transcript.


Assuntos
Proteínas Fúngicas/fisiologia , Iniciação Traducional da Cadeia Peptídica , Fatores de Iniciação de Peptídeos/fisiologia , Capuzes de RNA/genética , RNA Fúngico/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Endorribonucleases/metabolismo , Exorribonucleases/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Meia-Vida , Substâncias Macromoleculares , Mutação , Fatores de Iniciação de Peptídeos/deficiência , Fatores de Iniciação de Peptídeos/genética , Proteínas de Ligação ao Cap de RNA , Saccharomyces cerevisiae/metabolismo
17.
Trends Biotechnol ; 17(7): 297-302, 1999 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-10370237

RESUMO

Physical mapping has been rediscovered as an important component of large-scale sequencing projects. Restriction maps provide landmark sequences at defined intervals, and high-resolution restriction maps can be assembled from ensembles of single molecules by optical means. Such optical maps can be constructed from both large-insert clones and genomic DNA, and are used as a scaffold for accurately aligning sequence contigs generated by shotgun sequencing.


Assuntos
Óptica e Fotônica , Análise de Sequência de DNA/métodos , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA/genética , Genoma Humano , Humanos , Dados de Sequência Molecular
18.
Biochem Biophys Res Commun ; 258(1): 102-8, 1999 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-10222242

RESUMO

The dynamics of individual DNA molecules in a thin gel were studied with fluorescence microscopy. Driven by an electric field, molecules hooked around isolated obstacles and became extended. By analyzing molecular images, we identified the reptation tube and primitive chain. When the field was turned off, the molecules relaxed. The relaxation time tau1 and primitive chain length at equilibrium depend on N, the size of the molecule in base pairs, consistently with reptation theory. Using five yeast chromosomal DNAs ranging in size from 245 kb to 980 kb, we found that: These results constitute a way of sizing individual DNA molecules by imaging rather than by gel electrophoresis.


Assuntos
DNA Fúngico/química , Conformação de Ácido Nucleico , Cromossomos Fúngicos , Microscopia de Fluorescência
19.
Genome Res ; 9(2): 175-81, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10022982

RESUMO

Detailed restriction maps of microbial genomes are a valuable resource in genome sequencing studies but are toilsome to construct by contig construction of maps derived from cloned DNA. Analysis of genomic DNA enables large stretches of the genome to be mapped and circumvents library construction and associated cloning artifacts. We used pulsed-field gel electrophoresis purified Plasmodium falciparum chromosome 2 DNA as the starting material for optical mapping, a system for making ordered restriction maps from ensembles of individual DNA molecules. DNA molecules were bound to derivatized glass surfaces, cleaved with NheI or BamHI, and imaged by digital fluorescence microscopy. Large pieces of the chromosome containing ordered DNA restriction fragments were mapped. Maps were assembled from 50 molecules producing an average contig depth of 15 molecules and high-resolution restriction maps covering the entire chromosome. Chromosome 2 was found to be 976 kb by optical mapping with NheI, and 946 kb with BamHI, which compares closely to the published size of 947 kb from large-scale sequencing. The maps were used to further verify assemblies from the plasmid library used for sequencing. Maps generated in silico from the sequence data were compared to the optical mapping data, and good correspondence was found. Such high-resolution restriction maps may become an indispensable resource for large-scale genome sequencing projects.


Assuntos
Cromossomos/genética , Plasmodium falciparum/genética , Mapeamento por Restrição/métodos , Animais , DNA de Protozoário/análise , Processamento de Imagem Assistida por Computador , Microscopia de Fluorescência , Mapeamento Físico do Cromossomo
20.
Biochem Biophys Res Commun ; 254(2): 466-73, 1999 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-9918862

RESUMO

Single molecule approaches to the characterization of biochemical systems offer an intrinsically simple and direct approach to address difficult, previously unyielding problems. Optically based approaches have recently been used to construct high resolution, ordered restriction maps from a variety of clone types. Advancements in surface technologies have enabled the reliable elongation and fixation of large DNA molecules onto specially derivatized substrates with retention of biochemical accessibility. In this study, the addition of fluorescently labeled nucleotides to surface-mounted DNA molecules by the action of DNA polymerase I is investigated using fluorescence microscopy to image individual template molecules. Molecules undergoing nick translation and containing only a few fluorochromes are readily imaged. These novel results suggest that surface-bound molecules may serve as a substrate for a broad range of enzymatic actions, and may offer new routes to analysis when coupled to advanced imaging techniques.


Assuntos
DNA Polimerase I/metabolismo , DNA Viral/biossíntese , Bacteriófago lambda/genética , Sequência de Bases , Primers do DNA , DNA Viral/genética , Corantes Fluorescentes , Cinética , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Moldes Genéticos
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