RESUMEN
We describe the application of Extreme Value Statistics to the analysis of discrete species that possess distinguishable properties (fluorescence wavelength, fluorescence intensity, light scattering, etc.) as they cross a well-defined observation/probe region. Time-gated selection and extreme value data analysis result in increased resolution in analytical determinations. When only the data corresponding to the smallest crossing times are selected for analysis, the width of the diffusion band decreases for the measured parameter. The molecules with the smallest crossing times diffuse preferentially along the flow direction. A Monte Carlo technique and the probability density function (pdf) for a freely diffusing species are used to generate data streams to provide a theoretical basis for the aforementioned phenomenon. These calculations are included to characterize the effect of the average flow rate and the diffusion constant. We have also included a procedure for extracting the normal diffusion constant (D) from the Extreme Value Distribution. In contrast to standard flow analysis, which requires long path lengths, our approach is particularly suited for measurements in picolitre and nanolitre volumes and provides another dimension to single-molecule measurements in cellular size volumes. We believe that this is a general phenomenon that depends upon the details of the pdf, which can be complex.
Asunto(s)
Espectrometría de Fluorescencia/métodos , Difusión , Nanopartículas del Metal/química , Método de Montecarlo , Plata/química , Factores de TiempoRESUMEN
A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating Bacillus anthracis from B. cereus and B. thuringiensis. A worldwide collection of 25 B. anthracis isolates showed high-profile homology, and these isolates were unambiguously distinguished from B. cereus and B. thuringiensis isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.
Asunto(s)
Bacillus anthracis/clasificación , Bacillus cereus/clasificación , Bacillus thuringiensis/clasificación , Técnicas de Tipificación Bacteriana , ADN Bacteriano/análisis , Bacillus anthracis/genética , Bacillus cereus/genética , Bacillus thuringiensis/genética , Análisis por Conglomerados , ADN Bacteriano/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Electroforesis en Gel de Campo Pulsado , Genotipo , FilogeniaRESUMEN
Rapid binding kinetics of SYTOX Orange stain with double-stranded DNA (dsDNA) was revealed on the DNA fragment sizing flow cytometer. We demonstrated for the first time that the dye molecules could be adsorbed onto the capillary surface and native DNA fragments can be dynamically stained while passing through the capillary. High-quality burst size distribution histograms were obtained for DNA samples analyzed immediately after staining, dilution, or mixing. These observations indicated that rapid interactions exist between SYTOX Orange dye molecules and dsDNA. A stopped-flow fluorescence apparatus was set up to capture the fast association traces of intercalating dyes binding to dsDNA. Kinetic equations were derived to fit the association curves for determination of association rates and to model the dynamic staining, dilution, and mixing processes of DNA samples stained with intercalating dyes. The measured association rates for both SYTOX Orange and PicoGreen stains intercalating into dsDNA were on the order of 10(8) M-1 s-1, suggesting a diffusion-controlled process. Simulations indicate that reequilibration can be reached in seconds upon staining, dilution, or mixing. Insight into the kinetics of DNA binding dyes will help implement efficient sample-handling practices in DNA analysis, including DNA fragment sizing flow cytometry.
Asunto(s)
ADN/análisis , Sitios de Unión , ADN/química , Citometría de Flujo/métodos , Cinética , Compuestos Orgánicos/química , Tamaño de la Partícula , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/métodos , Coloración y Etiquetado , Relación Estructura-Actividad , Factores de TiempoRESUMEN
We demonstrate the use of technology developed for optical mapping to acquire DNA fingerprints from single genomes for the purpose of discrimination and identification of bacteria and viruses. Single genome fingerprinting (SGF) provides not only the size but also the order of the restriction fragments, which adds another dimension to the information that can be used for discrimination. Analysis of single organisms may eliminate the need to culture cells and thereby significantly reduce analysis time. In addition, samples containing mixtures of several organisms can be analyzed. For analysis, cells are embedded in an agarose matrix, lysed, and processed to yield intact DNA. The DNA is then deposited on a derivatized glass substrate. The elongated genome is digested with a restriction enzyme and stained with the intercalating dye YOYO-1. DNA is then quantitatively imaged with a fluorescence microscope and the fragments are sized to an accuracy >or=90% by their fluorescence intensity and contour length. Single genome fingerprints were obtained from pure samples of adenovirus, from bacteriophages lambda and T4 GT7, and from a mixture of the three viral genomes. SGF will enable the fingerprinting of uncultured and unamplified samples and allow rapid identification of microorganisms with applications in forensics, medicine, public health, and environmental microbiology.
Asunto(s)
Dermatoglifia del ADN/métodos , Genoma Viral , Calibración , ADN Viral/análisis , ADN Viral/genética , Fluorescencia , Tamaño de la PartículaRESUMEN
BACKGROUND: The measurement of physical properties from single molecules has been demonstrated. However, the majority of single-molecule studies report values based on relatively large data sets (e.g., N > 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters. METHODS: Monte Carlo simulations and multinomial analysis, dependent on quantifiable experimental parameters, were used to determine the minimum number of single-molecule measurements required to produce an accurate estimate of a population mean. Simulation results were applied to the fluorescence-based sizing of DNA fragments by ultrasensitive flow cytometry (FCM). RESULTS: Our simulations show, for an analytical technique with a 10% CV, that the average of as few as five single-molecule measurements would provide a mean value within one SD of the population mean. Additional simulations determined the number of measurements required to obtain the desired number of replicates for each subpopulation within a mixture. Application of these results to flow cytometry data for lambda/HindIII and S. aureus Mu50/SmaI DNA digests produced accurate DNA fingerprints from as few as 98 single-molecule measurements. CONCLUSIONS: A surprisingly small number of single-molecule measurements are required to obtain a mean measurement descriptive of a normally-distributed parent population.
Asunto(s)
Bacteriófago lambda/química , Dermatoglifia del ADN/estadística & datos numéricos , Fragmentación del ADN , ADN/análisis , Citometría de Flujo/estadística & datos numéricos , Staphylococcus aureus/química , Dermatoglifia del ADN/métodos , Citometría de Flujo/métodos , Método de Montecarlo , Reproducibilidad de los ResultadosRESUMEN
The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% +/- 2%) and FCM (4% +/- 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSD(PFGE) ] = 3% +/- 2% and RSD(FCM) = 1.2% +/- 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional band-based methods.
Asunto(s)
ADN Bacteriano/química , Electroforesis en Gel de Campo Pulsado/métodos , Citometría de Flujo/métodos , Técnicas Bacteriológicas/estadística & datos numéricos , Dermatoglifia del ADN , ADN Bacteriano/aislamiento & purificación , Electroforesis en Gel de Campo Pulsado/estadística & datos numéricos , Citometría de Flujo/estadística & datos numéricos , Peso Molecular , Staphylococcus aureus/químicaRESUMEN
This paper outlines the first use of SYTOX Orange, SYTO 82 and SYTO 25 nucleic acid stains for on-column staining of double-stranded DNA (dsDNA) fragments separated by capillary electrophoresis (CE). Low-viscosity, replaceable poly(vinylpyrrolidone) (PVP) polymer solution was used as the sieving matrix on an uncoated fused-silica capillary. The effects of PVP concentration, electric field strength, and incorporated nucleic acid stain concentrations on separation efficiency were examined for a wide range of DNA fragment sizes. Our study was focused on using nucleic acid stains efficiently excitable at a wavelength of 532 nm. Among the five tested nucleic acid stains, SYTOX Orange stain was shown to have the best sensitivity for dsDNA detection by CE. About a 500-fold lower detection limit was obtained compared to commonly used ethidium bromide and propidium iodide. SYTOX Orange stain also provided a wide linear dynamic range for direct DNA quantitation with on-line CE detection. Use of SYTOX Orange stain can greatly improve the measurement of DNA fragments by CE, which will enable an expanded set of applications in genomics and diagnostics.