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1.
Clin Genet ; 94(6): 564-568, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30084132

RESUMO

Biparental/androgenetic mosaicism is a rarely diagnosed condition in humans. It is typically ascertained prenatally on the basis of placental mesenchymal dysplasia. Fetal outcome can range from demise due to intrauterine growth retardation to term delivery. Most of the published cases of liveborns represent females that are either completely normal or have features of Beckwith-Wiedemann syndrome. Only two healthy liveborn males with mosaicism detected in the placenta have been described to date. Here, we report another liveborn male with hepatic mesenchymal hamartoma, soft tissue overgrowth on his right fifth toe, hemangiomas over his chest, right buttock and foot, anemia, thrombocytopenia and congenital hypothyroidism with biparental/androgenetic mosaicism detected in the toe mass in addition to the placenta. This new case adds to the existing literature of individuals with biparental/androgenetic mosaicism and expands the range of clinical presentations that may be seen in male patients with this condition. This study also illustrates the important use of single-nucleotide polymorphism microarray in conjunction with short-tandem repeat analysis on affected tissue to provide a diagnosis for patients with features of overgrowth and prior, non-diagnostic, genetic analyses of their peripheral blood.


Assuntos
Anormalidades Congênitas/diagnóstico , Anormalidades Congênitas/genética , Mosaicismo , Placenta/metabolismo , Placenta/patologia , Biópsia , Bandeamento Cromossômico , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Testes Genéticos , Variação Genética , Humanos , Hibridização in Situ Fluorescente , Lactente , Hepatopatias/diagnóstico , Hepatopatias/genética , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único , Gravidez
2.
Dev Cell ; 1(4): 579-86, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11703947

RESUMO

During Drosophila development, the Jun N-terminal kinase signal transduction pathway regulates morphogenetic tissue closure movements that involve cell shape changes and reorganization of the actin cytoskeleton. We analyzed the genome-wide transcriptional response to activation of the JNK pathway in the Drosophila embryo by serial analysis of gene expression (SAGE) and identified loci encoding cell adhesion molecules and cytoskeletal regulators as JNK responsive genes. The role of one of the upregulated genes, chickadee (chic), encoding a Drosophila profilin, in embryogenesis was analyzed genetically. chic-deficient embryos fail to execute the JNK-mediated cytoskeletal rearrangements during dorsal closure. This study demonstrates a transcriptional mechanism of cytoskeletal regulation and establishes SAGE as an advantageous approach for genomic experiments in the fruitfly.


Assuntos
Proteínas Contráteis , Drosophila melanogaster/embriologia , Proteínas Quinases JNK Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Transdução de Sinais/genética , Actinas/metabolismo , Animais , Adesão Celular/fisiologia , Citoesqueleto/metabolismo , Proteínas de Drosophila , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento , MAP Quinase Quinase 4 , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Profilinas , Transcrição Gênica/fisiologia
3.
J Hosp Infect ; 65(2): 124-30, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17174445

RESUMO

The aim of this study was to obtain data concerning the incidence, reporting and follow-up of occupational exposure to blood or other body fluids (OEB). A questionnaire was distributed to employees and medical students (N=787) and official reports of OEB during the year 2003 (N=203) and their consequent follow-up (N=100) were evaluated. The percentages of needlestick injuries were 29.5% for students and 22.5% for employees. Incidence rates per 1000 employee days were 0.61 for needlestick injuries or sharp object injuries and 0.27 for mucocutaneous exposure to body fluids. The mean rate of underreporting was approximately 45%. Contrary to expectations, only 4.3% of nurses and 3.9% of doctors officially reported an OEB in 2003. The number of persons who did not attend for a serological test increased during the follow-up period. Considering all documented test results, 35 out of 100 affected persons were lost to follow-up due to default of appearance. As a consequence, the employer should provide safety devices and enforce didactical interventions with practical training and incident reporting. Periodical occupational health medicals, including serological testing, should be mandatory for all employees, including medical students and student nurses. To increase compliance after OEB, a short follow-up period using improved laboratory tests requires further discussion.


Assuntos
Pessoal de Saúde/estatística & dados numéricos , Ferimentos Penetrantes Produzidos por Agulha/epidemiologia , Exposição Ocupacional/efeitos adversos , Estudantes de Medicina/estatística & dados numéricos , Seguimentos , Alemanha/epidemiologia , Hospitais Universitários , Humanos , Incidência , Ferimentos Penetrantes Produzidos por Agulha/sangue , Ferimentos Penetrantes Produzidos por Agulha/virologia , Exposição Ocupacional/estatística & dados numéricos , Estudos Prospectivos , Inquéritos e Questionários
4.
Mol Cell Biol ; 8(10): 4416-24, 1988 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-3185555

RESUMO

The chicken erythrocyte anion transport protein (band 3 of the erythrocyte cytoskeleton) is a central component taking part in two widely divergent functions of erythroid cells; it is a primary determinant of cytoskeletal architecture and responsible for electroneutral Cl-/HCO3- exchange across the plasma membrane. To analyze interesting aspects of the developmental regulation of this gene, we have cloned the cDNA and genomic counterparts of the erythroid-specific anion transport protein. We show that a single genetic locus for band 3 encodes two different erythroid cell-specific mRNAs, with different translational initiation sites, which predict polypeptides of sizes very close to those observed in vivo. In vitro translation and immune precipitation of synthetic mRNA derived from one putative fully encoding cDNA clone demonstrate that this clone gives rise to a protein which is identical in size and antigenicity to bona fide chicken erythroid band 3.


Assuntos
Proteína 1 de Troca de Ânion do Eritrócito/genética , Galinhas/genética , Membrana Eritrocítica/fisiologia , RNA Mensageiro/genética , Sequência de Aminoácidos , Animais , Proteína 1 de Troca de Ânion do Eritrócito/imunologia , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA/genética , Genes , Dados de Sequência Molecular , Testes de Precipitina , Biossíntese de Proteínas , Transcrição Gênica
5.
Phys Med Biol ; 61(17): N441-60, 2016 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-27499388

RESUMO

The hybrid technology cell-fluorescent ion track hybrid detector (Cell-Fit-HD) enables the investigation of radiation-related cellular events along single ion tracks on the subcellular scale in clinical ion beams. The Cell-Fit-HD comprises a fluorescent nuclear track detector (FNTD, the physical compartment), a device for individual particle detection and a substrate for viable cell-coating, i.e. the biological compartment. To date both compartments have been imaged sequentially in situ by confocal laser scanning microscopy (CLSM). This is yet in conflict with a functional read-out of the Cell-Fit-HD utilizing a fast live-cell imaging of the biological compartment with low phototoxicity on greater time scales. The read-out of the biological from the physical compartment was uncoupled. A read-out procedure was developed to image the cell layer by conventional widefield microscopy whereas the FNTD was imaged by CLSM. Point mapping registration of the confocal and widefield imaging data was performed. Non-fluorescent crystal defects (spinels) visible in both read-outs were used as control point pairs. The accuracy achieved was on the sub-µm scale. The read-out procedure by widefield microscopy does not impair the unique ability of spatial correlation by the Cell-Fit-HD. The uncoupling will enlarge the application potential of the hybrid technology significantly. The registration allows for an ultimate correlation of microscopic physical beam parameters and cell kinetics on greater time scales. The method reported herein will be instrumental for the introduction of a novel generation of compact detectors facilitating biodosimetric research towards high-throughput analysis.


Assuntos
Fenômenos Fisiológicos Celulares , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Radiometria/instrumentação , Radiometria/métodos , Células A549 , Óxido de Alumínio/química , Sobrevivência Celular , Fluorescência , Humanos , Transferência Linear de Energia , Microscopia Confocal/instrumentação
6.
FEBS Lett ; 233(2): 432-6, 1988 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-3289973

RESUMO

The protocol for Sanger dideoxy chain termination reactions in DNA sequencing is tedious and prone to errors due to the repetitive character of the pipetting steps. An industrial robot, with the addition of a few simple parts, was programmed to automate the dideoxy sequencing reactions. The system is set up in a short time for routine operation and it is faster and more reliable than a human operator. It is flexible and allows variations and optimization of the standard procedure. Disposable microtiter plates at a controlled temperature are used. In one reaction cycle (about 50 min) up to 48 templates are processed. Up to 450 bases were resolved in automated DNA sequencing on samples prepared by the robot. The protocol is applicable to fluorescent as well as to radioactive labeling.


Assuntos
Sequência de Bases , DNA Viral , Autoanálise/instrumentação , Autoanálise/métodos , Colífagos , DNA Viral/isolamento & purificação , Escherichia coli
7.
Biotechniques ; 17(2): 302, 304, 306 passim, 1994 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-7980933

RESUMO

Recently available thermostable DNA polymerases result in enhanced resolution and accuracy compared to thermal enzymes used previously in fluorescent cycle sequencing. These new enzymes produce less variations in peak intensities, enhance gel resolution and are less sensitive to unspecific termination caused by either DNA structure or impurities in the DNA preparation. Optimization of nucleotide ratios and the usage of high concentrations of detergents in the sequencing reaction result in sequence readings up to 1000 bases and improve overall reliability of the sequencing protocol; this works successfully in about 90% of cases.


Assuntos
Análise de Sequência de DNA/métodos , DNA Polimerase Dirigida por DNA , Fluorescência , Temperatura Alta
8.
Biotechniques ; 33(3): 620-8, 630, 2002 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-12238772

RESUMO

Gene expression profiling by DNA microarrays has found wide application in many fields of biomedical research. The protocols for this technique are not yet standardized, and for each given step in microarray analysis a number of different protocols are in use. As a consequence, results obtained in different laboratories can be difficult to compare. Of particular importance in this respect are the methods for the preparation of fluorescent cDNA probes that should quantitatively reflect the abundance of different mRNAs in the two samples to be compared. Here we systematically evaluate and compare five different published and/or commercial principles for the synthesis offluorescently labeled probes for microarray analysis (direct labeling, 77 RNA polymerase amplification, aminoallyl labeling, hapten-antibody enzymatic labeling, and 3-D multi-labeled structures). We show that individual labeling methods can significantly influence the expression pattern obtained in a microarray experiment and discuss the respective benefits and limitations of each method.


Assuntos
Sondas de DNA/síntese química , Corantes Fluorescentes/síntese química , Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , DNA Complementar/química , Células HeLa/fisiologia , Humanos , Deficiências de Ferro , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Coloração e Rotulagem/métodos
9.
Biotechniques ; 18(4): 688-97, 1995 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-7541216

RESUMO

We have identified additional criteria for the walking primer design that improve the success rate of automated fluorescent DNA sequencing using the internal labeling technique and T7 DNA polymerase. These criteria resulted from the evaluation of over 220 sequences generated with walking primers and fluorescein-15-dATP as internal label in the course of the European Community (EC) yeast genome sequencing project. In this project primers were designed using standard commercial software. Intensities of sequencing signals varied over a broad range from very strong to very weak, depending on the primers used. This led us to evaluate primer performance relative to (i) the template sequence immediately downstream of the primer binding site and (ii) the primer sequence itself. Our experiments show that the position of the first labeled dATP to be incorporated downstream of the primer into the growing strand is substantial for the signal intensity of the sequence. The closer to the primer that the first 'A' is incorporated, the stronger the peak intensities are. An additional feature of sequencing with native T7 DNA polymerase is its ability to remove a 3'-terminal 'A' of the primer by the 3'-->5' exonuclease activity and to exchange the nucleotide with a labeled dATP by the polymerase activity.


Assuntos
Primers do DNA/genética , DNA Polimerase Dirigida por DNA , Nucleotídeos de Desoxiadenina , Fluoresceínas , DNA Polimerase Dirigida por RNA/genética , Análise de Sequência de DNA/métodos , Sítios de Ligação , Exonucleases/genética
10.
Biotechniques ; 15(4): 714-21, 1993 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-8251174

RESUMO

Low-redundancy automated DNA sequencing by primer walking is described. T7 DNA polymerase is used together with computer-selected walking primers and fluorescein-dATP as internal label to sequence large plasmids or cosmids directly on a standard DNA sequencer with an error rate below 1% up to 500 bases (in the unedited raw data). The low error rate allows efficient sequencing with low (2-3 times) redundancy. Plasmid subclones covering 20 kb of a cosmid insert were sequenced with an overall redundancy of 2.7 in the course of the European community Saccharomyces cerevisiae genome sequencing project. Neighboring plasmid subclones were linked by direct cosmid sequencing. Sets of ten walking primers are synthesized on the EMBL multiple segmental DNA synthesizer at low costs and used for sequencing with greater than 95% efficiency. The accuracy of the directed approach is improved by simultaneous walking on both strands by designing two primers in opposite directions in the same starting region. One primer is used to confirm sequence data on the opposite strand, and the other primer to obtain new sequence data.


Assuntos
Passeio de Cromossomo , Primers do DNA , Genes Fúngicos , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA/métodos , Autoanálise , Cosmídeos , Primers do DNA/economia , DNA Polimerase Dirigida por DNA , Nucleotídeos de Desoxiadenina , Fluoresceína , Fluoresceínas , Plasmídeos
11.
J Biotechnol ; 41(2-3): 121-9, 1995 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-7654345

RESUMO

An efficient low redundancy DNA sequencing strategy should allow high accuracy determination of the consensus sequence on both strands of a DNA fragment from a minimal number of sequencing reactions with minimal overlap. At EMBL we developed a directed strategy for cosmid-scale sequencing based on primer walking, whereas most other sequencing projects of this scale rely on the random 'shotgun' strategy. In our strategy, highly accurate raw data are obtained from automated double-stranded Sanger dideoxy sequencing with inexpensive walking primers (8 to 10 $ per primer), T7 DNA polymerase and internal labelling by fluorescein-15- dATP on A.L.F. DNA sequencers (Pharmacia Biotech). The use of 60-cm long glass plates enables reading length of up to 1000 bases. Comparing various random and directed sequencing strategies in the course of the European Community yeast genome sequencing project on cosmids from chromosomes IX, XI and XV, primer walking was found to be the strategy resulting in the lowest possible redundancy of 2.6 to 2.8. Future development of the sequencing strategy is based on the new EMBL 2-dye sequencing device for simultaneous sequencing on both strands, and implementation of an initial limited random sequencing phase to reduce the number of walking primers required by a factor of 3, while still maintaining a low redundancy of approx. 3.


Assuntos
Sequência de Bases , DNA/química , DNA/genética , Bases de Dados Factuais , Animais , Biotecnologia/métodos , Cromossomos Fúngicos , DNA/biossíntese , Primers do DNA , Dados de Sequência Molecular , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico
12.
J Biochem Biophys Methods ; 45(1): 65-74, 2000 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-10899391

RESUMO

High quality lane-tracking of gel images is the first task, and thus a prerequisite, for successful trace processing and base-calling of DNA sequencing slab gels. In most approaches, it is based on statistical calculations, for instance variance and co-variance analysis between neighboring pixel columns in the image. On the basis of these statistical calculations, Kohonen's self-organization neural network model was introduced. We have found that, using several well-structured input data, Kohonen's self-organization neural network model can be trained to fulfill our task of lane-tracking. Furthermore, the quality of lane-tracking could be improved compared to algorithmic approaches.


Assuntos
Redes Neurais de Computação , Análise de Sequência de DNA/métodos , Algoritmos , Simulação por Computador , Eletroforese em Gel de Poliacrilamida/métodos , Modelos Teóricos
13.
J Biochem Biophys Methods ; 13(6): 315-23, 1986 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-3559035

RESUMO

A method and instrument for automated DNA sequencing without radioactivity have been developed. In spite of the success with radioactive labels there are drawbacks attached to the technique, such as hazards in the handling, storage and disposal of radioactive materials, and the considerable cost of the radiolabelled nucleoside triphosphates. In addition, there is deterioration of sample quality with time. A sulphydryl containing M13 sequencing primer has been synthesised and subsequently conjugated with tetramethylrhodamine iodoacetamide. The fluorescent primer is used to generate a nested set of fluorescent DNA fragments. The fluorescent bands are excited by a laser and detected in the gel (detection limit about 0.1 fmol per band) during electrophoresis, and sequence data from the four tracks are transferred directly into a computer. Standard gels, 200 mm wide with 20 sample slots have also been used. The device contains no moving parts. At present 250-300 bases can be read in 6 h. The system is capable of single base resolution at a fragment length of at least 400 bases.


Assuntos
DNA/análise , Autoanálise , Sequência de Bases , Eletroforese em Gel de Poliacrilamida , Corantes Fluorescentes , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética
14.
J Biochem Biophys Methods ; 20(1): 47-52, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2630585

RESUMO

Novel Sanger dideoxy sequencing with only one fluorescent dye label for the four bases of one clone and sequence determination in two lanes on polyacrylamide gel is presented, loading A greater than G in one lane and T greater than C in the other. Sequencing reactions for the two bases in each lane are carried out in one tube. At present the ratio of ddATP:ddGTP and ddTTP:ddCPT is set to 5:1 in the two tubes. Distinction between the two bases in one lane is done by comparing the different magnitudes of the peaks. This method increases the capacity since more clones may be run simultaneously on one gel, while keeping the reliability and simplicity that comes with the use of only one fluorescent dye for the four bases of one clone. At present about 200 bases are determined with the one-dye two-lane method on the EMBL's automated fluorescent DNA sequencer, using T7 DNA polymerase. The error rate in the deduced sequence is about 1%. The technique is used for the determination of overlaps in mapping projects. In principle, it is possible to determine the sequence with one dye in only one lane on the gel by choosing the proper ddNTP ratios for all four bases, carrying out reactions in one tube and applying the product in one lane, but the error rate for this one-lane method seems too high at present and further improvements in the uniformity of peaks obtainable with the T7 DNA polymerase or other enzymes are required.


Assuntos
Sequência de Bases , DNA , DNA Polimerase Dirigida por DNA , Eletroforese em Gel de Poliacrilamida , Métodos , Dados de Sequência Molecular
15.
DNA Seq ; 1(1): 63-71, 1990.
Artigo em Inglês | MEDLINE | ID: mdl-2132960

RESUMO

A new general method for sequencing fluorescently labelled DNA by chemical degradation has been developed. It is based on the observation that fluorescein attached via a mercaptopropyl or aminopropyl linker arm to the 5'-phosphate of an oligonucleotide is stable during the reactions commonly used in chemical cleavage procedures. DNA to be degraded is first enzymatically synthesized in vitro by annealing and extending a fluorescently labelled primer thereby introducing the fluorescent label at the 5'-end of the fragment. The newly synthesized fluorescently labelled DNA is then chemically degraded using: (a) a set of four different cleavage reactions; or (b) only one reaction comprising methylation of G-residues followed by a partial cleavage with piperidine in the presence of sodium chloride. The fluorescent degradation products are loaded on either four lanes or one lane of the gel, respectively, and the emitted fluorescence detected online during electrophoresis. In the 'four reactions/four lanes' method 200-350 bp (base pairs) can be read from the labelled end. The 'one reaction/one lane' method, in which the nucleotide sequence is determined by measuring different signal intensities following the rule G greater than A greater than C greater than T, currently yields around 100-200 bp of sequence per sample.


Assuntos
Sequência de Bases , DNA/genética , Automação , DNA/química , Corantes Fluorescentes , Técnicas Genéticas , Dados de Sequência Molecular , Estrutura Molecular
16.
Oncogene ; 29(10): 1519-30, 2010 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-20023695

RESUMO

The Ras oncogene is known to activate three major MAPK pathways, ERK, JNK, p38 and exert distinct cellular phenotypes, that is, apoptosis and invasion through the Ras-MKK3-p38-signaling cascade. We attempted to identify the molecular targets of this pathway that selectively govern the invasive phenotype. Stable transfection of NIH3T3 fibroblasts with MKK3(act) cDNA construct revealed similar p38-dependent in vitro characteristics observed in Ha-Ras(EJ)-transformed NIH3T3 cells, including enhanced invasiveness and anchorage-independent growth correlating with p38 phosphorylation status. To identify the consensus downstream targets of the Ras-MKK3-p38 cascade involved in invasion, in vitro invasion assays were used to isolate highly invasive cells from both, MKK3 and Ha-Ras(EJ) transgenic cell lines. Subsequently a genome-wide transcriptome analysis was employed to investigate differentially regulated genes in invasive Ha-Ras(EJ)- and MKK3(act)-transfected NIH3T3 fibroblasts. Using this phenotype-assisted approach combined with system level protein-interaction network analysis, we identified FOXM1, PLK1 and CDK1 to be differentially regulated in invasive Ha-Ras(EJ)-NIH3T3 and MKK3(act)-NIH3T3 cells. Finally, a FOXM1 RNA-knockdown approach revealed its requirement for both invasion and anchorage-independent growth of Ha-Ras(EJ)- and MKK3(act)-NIH3T3 cells. Together, we identified FOXM1 as a key downstream target of Ras and MKK3-induced cellular in vitro invasion and anchorage-independent growth signaling.


Assuntos
Fatores de Transcrição Forkhead/genética , Perfilação da Expressão Gênica , MAP Quinase Quinase 3/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas ras/genética , Animais , Western Blotting , Adesão Celular , Movimento Celular , Flavonoides/farmacologia , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/metabolismo , Imidazóis/farmacologia , MAP Quinase Quinase 3/antagonistas & inibidores , MAP Quinase Quinase 3/metabolismo , Camundongos , Células NIH 3T3 , Análise de Sequência com Séries de Oligonucleotídeos , Piridinas/farmacologia , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/metabolismo
18.
Nucleic Acids Res ; 15(11): 4593-602, 1987 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-3588303

RESUMO

A simple system has been designed enabling ultrasensitive on-line detection of fluorescently labelled macromolecules, e.g. nucleic acids, proteins and peptides during electrophoretic separations in gels. An important application is the automated DNA sequence determination without radioactivity. Drying of gels, film exposure and handling are not necessary. A sulphydryl containing M13 sequencing primer has been synthesised and end-labelled in a reaction with fluorescein iodoacetamide. This is then used in the dideoxy reactions. In particular no moving parts or complicated software are required for data collection and analysis. Compared to our first automated device detection sensitivity has been improved by a factor of thirty to about 3 X 10(-18) mol per band. The resolution has increased to about 400 bases in 5 hours, with the possibility to read up to about 500 bases when they are properly labelled. Gels shorter than 20 cm may be used for resolution of about 300 bases. The single gel system may be upgraded for simultaneous running and reading of six or ten sequencing samples.


Assuntos
Autoanálise/métodos , Sequência de Bases , DNA/análise , Eletroforese em Gel de Poliacrilamida/métodos , Fluoresceína , Fluoresceínas , Peptídeos/análise , Proteínas/análise , Espectrometria de Fluorescência
19.
Nucleic Acids Res ; 16(5): 2203-6, 1988 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-3357772

RESUMO

A non-radioactive sequencing of fluorescently labelled oligonucleotides by solid-phase chemical degradation is described. Although non-radioactive methods have been reported for the dideoxy chain termination technique, such a method has not yet been developed for the chemical degradation sequencing of DNA fragments. A 21-mer fluorescein labelled M13 sequencing primer was sequenced in an on-line automated system in about 30 minutes. The fluorescent dye and its bond to the oligonucleotide were stable during the chemical reactions used for the base specific degradations. As the sequence is determined on-line during electrophoresis, reloading and running 10 fragments simultaneously allows us to use one gel for sequencing of about 50 different oligonucleotides.


Assuntos
Automação , Sequência de Bases , Oligonucleotídeos , Fenômenos Químicos , Química , Corantes Fluorescentes
20.
Nucleic Acids Res ; 16(8): 3487-96, 1988 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-3375061

RESUMO

T7 DNA polymerase with chemically inactivated 3'-5' exo-nuclease activity, as well as unmodified T7 DNA polymerase, were used for sequencing by the dideoxy method in an automated system with fluorescence labelled primer and on-line detection of laser-excited reaction products. An analysis of signal intensity variations in the C track revealed that low C signals were usually preceded by a T in the sequence. This effect was modified by surrounding nucleotides. Signal intensities were more uniform with T7 polymerase than with the Klenow fragment of DNA polymerase I. Some sequences ambiguous with the Klenow enzyme could easily be evaluated with the T7 enzyme. One sequence could only be read by the unmodified T7 polymerase, while both the Klenow fragment and the chemically modified T7 enzyme gave uninterpretable data.


Assuntos
DNA Polimerase Dirigida por DNA , DNA/análise , Mapeamento de Nucleotídeos/métodos , Sequência de Bases , DNA Polimerase I , Fagos T/enzimologia
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