RESUMO
INTRODUCTION: Group A streptococci (GAS) is responsible of several human diseases ranging from mild infection to severe invasive toxin-mediated disease and post-infectious sequelae. Accordingly, a GAS surveillance program based on molecular techniques is advisable for its epidemiological control. Pulsed-field gel electrophoresis (PFGE) is the gold standard for GAS molecular subtyping, but a major disadvantage is the length of the procedure, which takes 1-3 days of work, minimum. The aim of this study was to develop a rapid and cost-effective procedure for PFGE subtyping of GAS isolates. METHODOLOGY: Different incubation times of GAS, immobilized in agarose miniplugs, in solutions containing lysozyme and/or mutanolysine followed by solutions with urea instead of proteinase K, were assayed. DNA was restricted with SmaI and the fingerprints were obtained in clamped homogeneous electric field (CHEF) chambers and minichambers. The modified procedure was used to subtype 22 GAS isolates. RESULTS: Intact DNA molecules of GAS immobilized in agarose miniplugs were prepared incubating the cells, in situ, with a solution containing lysozyme for 4 hours, followed by the incubation in a non-enzymatic solution with urea for 2 hours. SmaIDNA macrorestriction fragments were well resolved in 5 hours and 14 minutes by electrophoresis in a CHEF minichamber at 10 V/cm. This procedure for GAS DNA preparation was useful for fingerprinting GAS strains in the format of CHEF Mapper (BioRad). CONCLUSIONS: The procedure took 13 hours for GAS strains subtyping. Both sample preparation and electrophoresis in CHEF minichamber represent an economic alternative for performing massive epidemiological studies of this human pathogen.
Assuntos
DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado/economia , Eletroforese em Gel de Campo Pulsado/métodos , Ácidos Nucleicos Imobilizados/genética , Tipagem Molecular/economia , Tipagem Molecular/métodos , Streptococcus pyogenes/classificação , Custos e Análise de Custo , DNA Bacteriano/isolamento & purificação , Humanos , Ácidos Nucleicos Imobilizados/isolamento & purificação , Epidemiologia Molecular/métodos , Streptococcus pyogenes/genética , Fatores de TempoRESUMO
BACKGROUND: Apolipoprotein E (ApoE) ε4 genotype is the most clearly documented risk factor for Alzheimer's disease (AD). Epidemiological studies demonstrate an accelerated rate of progression to dementia and AD in patients with mild cognitive impairment (MCI). We assessed the ApoE allele and genotypes frequencies in Cuban patients with MCI. METHODS: We performed ApoE genotyping of 74 Cuban patients more than 65 years old. Cognitive assessments included the Mini-Mental State Examination (MMSE) and a cognitive battery for evaluating memory, attention, perception, and executive function. RESULTS: Cognitive impairments were characterized by amnesia and executive deficits in patients with MCI. The Apo ε4 allele frequency was 0.196 in patients with MCI, 10-fold higher than that in the controls. Patients carrying the ε4 allele exhibited poorer performance in MMSE and tests assessing executive function and short-term memory than noncarriers. CONCLUSIONS: The patients exhibited amnestic MCI multiple domains. Cognitive performance was worse in patients who carried the ApoE ε4 allele.
Assuntos
Apolipoproteínas E/genética , Disfunção Cognitiva/genética , Frequência do Gene/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Apolipoproteína E4/genética , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/epidemiologia , Cuba/epidemiologia , Feminino , Humanos , MasculinoRESUMO
We standardized the zinc-imidazole negative staining method for detecting chromosomal-sized DNA molecules separated by pulsed field minigel electrophoresis. The best experimental conditions were as follows: separating large DNA molecules in minigels of 0.4 cm thickness, further incubating them with 40 mM ZnSO4 solution, and finally incubating them with 0.1 and 2 M imidazole solutions successively. The lowest yeast cells/miniplug useful in DNA band detection was 3 x 10(7) cells, as occurred with ethidium bromide-stained minigel. Electrophoresis patterns were visualized as colorless bands contrasting against a white background after illuminating the minigel with white light. This negative staining method is nontoxic and preserves the chemical integrity of the DNA molecules.
Assuntos
DNA Fúngico/análise , Eletroforese em Gel de Ágar/métodos , Imidazóis , Coloração Negativa/métodos , Saccharomyces cerevisiae/química , ZincoRESUMO
We propose cost-effective protocols for preparing and resolving the PulseNet universal DNA size standard in contour-clamped homogeneous electric field (CHEF) minichambers. Intact DNA molecules were prepared with protease-free solutions, and electrophoresis separations of the DNA standards needed 5.5h, giving band pattern resolutions similar to those attained with the PulseNet protocols standardized in CHEF chambers.
Assuntos
DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado/métodos , Salmonella/genética , DNA Bacteriano/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Reprodutibilidade dos Testes , Salmonella/classificação , SorotipagemRESUMO
DNA molecules suitable for amplification by Polymerase Chain Reaction were obtained by immobilizing whole blood or isolated leukocytes and incubating the immobilized cells for one hour with the known non-enzymatic solution described for preparing intact DNA molecules for PFGE. Cell immobilization was done in agarose gels and punches of 1.2 mm of diameter had the amount of DNA needed for amplifying chromosomal and mitochondrial sequences, many times. The approach was successfully used in preparing DNA molecules from multiple samples in flat-bottom 96-well ELISA plates. The procedure is simple and does not demand special conditions for sample transportation or conservation; thus, it should be useful to collect and process samples under field conditions in epidemiological studies.
Assuntos
Técnicas Biossensoriais , Células Sanguíneas/metabolismo , DNA/sangue , DNA/isolamento & purificação , Sequência de Bases , Células Sanguíneas/química , Cromossomos Humanos/genética , DNA/genética , DNA Mitocondrial/genética , Eletroforese em Gel de Ágar , Humanos , Técnicas de Amplificação de Ácido Nucleico , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
DNA molecules of Vibrio cholerae and Aeromonas species were prepared by incubating immobilized cells for 4 and 2 h, respectively, with a nonenzymatic solution that contains chemical reagents only (NDSUPlus). This method gave results as reproducible as the enzymatic one that uses proteinase K, and rendered DNA molecules suitable for fingerprinting by mini-CHEF electrophoresis. As rapid DNA separations at high electric field are achieved in mini-CHEF chamber with low heat evolution, DNA restriction fragments were separated in 5 h at 10 V/cm in a single resolution window. Then, fragment separations in three resolution windows were done in 15 h. This time is shorter than the one needed by the large CHEF chamber for resolving fragments in a single resolution window. Three windows permitted to include larger numbers of restriction fragments in the calculation of isolate similarities. Both sample preparation and mini-CHEF electrophoresis may represent an alternative for performing massive epidemiological studies of V. cholerae and Aeromonas species.
Assuntos
Aeromonas/isolamento & purificação , Técnicas de Tipagem Bacteriana , DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado/métodos , Vibrio cholerae/isolamento & purificação , Aeromonas/genética , Impressões Digitais de DNA , DNA Bacteriano/química , Vibrio cholerae/genéticaRESUMO
The ability of Entamoeba histolytica TATA binding protein (EhTBP) to interact with different TATA boxes in gene promoters may be one of the key factors to perform an efficient transcription in this human parasite. In this paper we used several TATA variants to study the in vitro EhTBP DNA-binding activity and to determine the TATA-EhTBP dissociation constants. The presence of EhTBP in complexes formed by nuclear extracts (NE) and the TATTTAAA oligonucleotide, which corresponds to the canonical TATA box for E. histolytica, was demonstrated by gel-shift assays. In these experiments a single NE-TATTTAAA oligonucleotide complex was detected. Complex was retarded by anti-EhTBP Igs in supershift experiments and antibodies also recognized the cross-linked complex in Western blot assays. Recombinant EhTBP formed specific complexes with TATA variants found in E. histolytica gene promoters and other TATA variants generated by mutation of TATTTAAA sequence. The dissociation constants of recombinant EhTBP for TATA variants ranged between 1.04 (+/-0.39) x 10(-11) and 1.60 (+/-0.37) x 10(-10) m. TATTTAAA and TAT_ _AAA motifs presented the lowest KD values. Intriguingly, the recombinant EhTBP affinity for TATA variants is stronger than other TBPs reported. In addition, EhTBP is more promiscuous than human and yeast TBPs, probably due to modifications in amino acids involved in TBP-DNA binding.
Assuntos
Entamoeba histolytica/metabolismo , TATA Box/fisiologia , Proteína de Ligação a TATA-Box/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sequência Conservada , Variação Genética , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese , Regiões Promotoras Genéticas , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TATA Box/genética , Proteína de Ligação a TATA-Box/química , Proteína de Ligação a TATA-Box/genéticaRESUMO
We present a transversal alternating field electrophoresis chamber that allows modifiable inner widths to accommodate low- or high-throughput formats, with 7.8 cm opposite electrode separation and 30.4 cm electrode length. Removable slotted sheets divide the chamber into four smaller compartments, each one supporting a minigel of 3.85 cm in length and 7.1 cm in width. Replacements of slotted sheets with solid dielectric blocks with the sizes and shapes of compartments permit to occlude chamber compartments, changing from 4 to 1 the numbers of minigels per run, from 88 to 13 the maximum numbers of samples, and from 1688 to 422 mL the volume of buffer poured into the chamber. Saccharomyces cerevisiae chromosomes gave its characteristic DNA band pattern in all compartments, whereas migrations of DNA molecules are not affected by the occlusion of compartments.
Assuntos
Cromossomos Fúngicos/genética , DNA Fúngico/genética , Eletroforese/instrumentação , Saccharomyces cerevisiae/genética , DNA Fúngico/análise , Eletroforese/métodosRESUMO
In the present work, a comparative study of 5-FdUrd, thy-, and metabolic in vivo labeling methods for plasmid and chromosomal DNA in E. coli DH5alpha cells was performed in order to achieve the best thymidine substitution method by 5-BrdUrd. According to the colorimetric immunoenzymatic results, we found that the minimal detectable labeled DNA (MDLD) was 312pg with the 5-FdUrd and thy- methods for 5-BrdUrd labeled plasmid DNA. 5-BrdUrd replaced about 96% of the total thymidine by 5-FdUrd methods; for the thy- and metabolic labeling methods, the MDLD value was 1,25 ng for denatured 5-BrdUrd chromosomal DNA. Pyrimidine nucleoside analogues were also evaluated as immunochemical markers for their in vivo introduction into DNA.
Assuntos
Bromodesoxiuridina , DNA/análise , Escherichia coli/genética , Floxuridina , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , Colorimetria/métodos , DNA/metabolismo , Inibidores Enzimáticos , Escherichia coli/metabolismo , Técnicas Imunoenzimáticas , Plasmídeos/análise , Plasmídeos/biossíntese , Timidina/química , Timidina/genética , Timidilato Sintase/antagonistas & inibidores , Timidilato Sintase/metabolismoRESUMO
We redesigned contour-clamped homogeneous electric field (CHEF) circuitry to eliminate crossover distortion, to set identical potentials at electrodes of each equipotential pair and to drive pairs with transistors in emitter follower stages. An equipotential pair comprised the two electrodes set at the same potential to provide electric field homogeneity inside of the hexagonal array. The new circuitry consisted of two identical circuits, each having a resistor ladder, diodes and transistors. Both circuits were interconnected by diodes that controlled the current flow to electrodes when the array was energized in the 'A' or 'B' direction of the electric field. The total number of transistors was two-thirds of the total number of electrodes. Average voltage deviation from potentials expected at electrodes to achieve a homogeneous electric field was 0.06 V, whereas 0.44 V was obtained with another circuit that used transistors in push-pull stages. The new voltage clamp unit is cheap, generated homogeneous electric field, and gave reproducible and undistorted DNA band patterns.
Assuntos
Eletroforese em Gel de Campo Pulsado/instrumentação , DNA Mitocondrial/análise , Eletrodos , Eletrônica , Desenho de Equipamento , Saccharomyces cerevisiae/genéticaRESUMO
The fastest protocol for Pseudomonas aeruginosa subtyping by contour clamped homogeneous electric field (CHEF) electrophoresis takes around 20 h. It includes enzymatic sample preparation, DNA restriction and fragment separation. Here, P. aeruginosa cells embedded in agarose miniplugs were lysed and deproteinized by incubating the miniplugs for 30 min in a single nonenzymatic solution. DNA molecules were digested for 2 h with 5 U of XbaI, and fragments were separated in 4.96 h by miniCHEF electrophoresis at 10 V/cm. Total time for P. aeruginosa subtyping was 8 h. Control experiments included DNA preparation by enzymatic or nonenzymatic protocols, different times of DNA restriction and comparisons of DNA separations done by miniCHEF or CHEF electrophoresis. Both methods and chambers gave similar results, but the rapid nonenzymatic method and the miniCHEF gave them in less time. Cells grown in broth or on plates were useful for nonenzymatic DNA preparation. Thirteen P. aeruginosa isolates were successfully fingerprinted using the protocol described here.
Assuntos
Técnicas Bacteriológicas/métodos , DNA Bacteriano/isolamento & purificação , Eletroforese em Gel de Campo Pulsado/métodos , Pseudomonas aeruginosa/genética , DNA Bacteriano/classificação , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Fatores de TempoRESUMO
We report a study on the DNA organization in Entamoeba histolytica using a ribosomal DNA probe. The rDNA genes were found forming mers which were separated in a typical ladder pattern by pulse field electrophoresis. DNA rosette structures were visualized through electron microscopy in DNA eluted from bands recognized by the ribosomal probe. The in situ hybridization experiments using a DNA probe suggested that the rDNA genes are portioned between the nucleus and a cytoplasmic structure. These findings provide new data on DNA organization in E. histolytica and open the question concerning the presence of a novel organelle in this eukaryotic parasite
Assuntos
Animais , DNA Ribossômico/genética , DNA Ribossômico/ultraestrutura , Citoplasma/química , DNA de Protozoário/genética , DNA de Protozoário/ultraestrutura , Entamoeba histolytica/genética , Entamoeba histolytica/ultraestrutura , Hibridização In SituRESUMO
Se realizaron 40 mediciones de siluetas ventriculares correspondientes a 20 ventriculografías realizadas en forma consecutiva. Los procedimientos utilizados fueron la planimetría, el recuento visual de los milímetros cuadrados incluidos en la silueta ventricular y el uso de tableta gráfica y lápiz digitalizador. Este último procedimiento se realizó en 2 centros distintos. El análisis de varianza de 2 clasificaciones no encontró diferencias significativas entre los distintos procedimientos por lo que éstos pueden utilizarse indistintamente para los cálculos de la ventriculografía cuantitativa (AU)
Assuntos
Humanos , Ventrículos do CoraçãoRESUMO
Se realizaron 40 mediciones de siluetas ventriculares correspondientes a 20 ventriculografías realizadas en forma consecutiva. Los procedimientos utilizados fueron la planimetría, el recuento visual de los milímetros cuadrados incluidos en la silueta ventricular y el uso de tableta gráfica y lápiz digitalizador. Este último procedimiento se realizó en 2 centros distintos. El análisis de varianza de 2 clasificaciones no encontró diferencias significativas entre los distintos procedimientos por lo que éstos pueden utilizarse indistintamente para los cálculos de la ventriculografía cuantitativa