We report here on the genome sequence of Pasteurella multocida Razi 0002 of avian origin, isolated in Iran. The genome has a size of 2,289,036 bp, a GC content of 40.3%, and is predicted to contain 2,079 coding sequences.

Optical approaches for observing the dynamics of single molecules have required pico- to nanomolar concentrations of fluorophore in order to isolate individual molecules. However, many biologically relevant processes occur at micromolar ligand concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. We show that arrays of zero-mode waveguides consisting of subwavelength holes in a metal film provide a simple and highly parallel means for studying single-molecule dynamics at micromolar concentrations with microsecond temporal resolution. We present observations of DNA polymerase activity as an example of the effectiveness of zero-mode waveguides for performing single-molecule experiments at high concentrations.

The fabrication of fluidic channels with dimensions smaller than 1 microm is described and characterized in respect to their use for detection of individual DNA molecules. The sacrificial layer technique is used to fabricate these devices as it provides CMOS-compatible materials exhibiting low fluorescence background. It also allows creating microfluidics circuitry of submicrometer dimensions with great control. The small dimensions facilitate single molecule detection and minimize events of simultaneous passage of more than one molecule through the measurement volume. The behavior of DNA molecules inside these channels under an applied electrical field was first studied by fluorescence correlation spectroscopy using M13 double-stranded DNA. A linear relationship between the flow speed and applied electric field across the channel was observed. Speeds as high as 5 mm/s were reached, corresponding to only a few milliseconds of analysis time per molecule. The channels were then used to characterize a mixture of nine DNA fragments. Both the distribution and relative proportions of the individual fragments, as well as the overall concentration of the DNA sample, can be deduced from a single experiment. The amount of sample required for the analysis was approximately 10,000 molecules, or 76 fg. Other potential applications of these submicrometer structures for DNA analysis are discussed.

Optical nanostructures have enabled the creation of subdiffraction detection volumes for single-molecule fluorescence microscopy. Their applicability is extended by the ability to place molecules in the confined observation volume without interfering with their biological function. Here, we demonstrate that processive DNA synthesis thousands of bases in length was carried out by individual DNA polymerase molecules immobilized in the observation volumes of zero-mode waveguides (ZMWs) in high-density arrays. Selective immobilization of polymerase to the fused silica floor of the ZMW was achieved by passivation of the metal cladding surface using polyphosphonate chemistry, producing enzyme density contrasts of glass over aluminum in excess of 400:1. Yields of single-molecule occupancies of Ϸ30% were obtained for a range of ZMW diameters (70 -100 nm). Results presented here support the application of immobilized single DNA polymerases in ZMW arrays for long-read-length DNA sequencing.fluorescence ͉ metal passivation ͉ microscopy ͉ polyvinyl phosphonic acid ͉ single molecule N anofabrication techniques have enabled new approaches to interrogate individual biomolecules by fluorescence techniques (reviewed in refs. 1 and 2). The extremely small size scale of the associated devices results in a drastic illumination volume reduction, allowing single-molecule investigations to take place at fluorophore concentrations increased to biologically relevant levels. In addition to higher temporal resolution and higher signal-to-noise ratios, they also provide spatial resolution beyond diffraction-limited optics.The zero-mode waveguide (ZMW) is one such nanophotonic confinement structure often consisting of a circular hole in a metal cladding film on a solid transparent substrate (3). In conjunction with laser-excited fluorescence, they provide observation volumes on the order of zeptoliters (10 Ϫ21 l), three to four orders of magnitude smaller than far-field excitation volumes. Applications of circular ZMWs have included the detection of single-molecule DNA polymerase activity by using labeled nucleotides at micromolar concentrations (3), the study of -repressor oligomerization dynamics (4), two-color crosscorrelation to rapidly screen for DNA restriction enzyme activity (5), and diffusion analysis of labeled membrane proteins in lipid bilayers of model membranes and living cells (6-11). C-shaped apertures have been described to study DNA hybridization interactions (12).ZMW technology applications have been limited by the unavailability of selective immobilization methods to position molecules exclusively in the observation volume, immediately above the transparent ZMW floor. One approach to selective immobilization exploits a feature inherent in the ZMW architecture. The transparent substrate and metal cladding are made of different materials, opening the possibility for a selective derivatization that will direct protein adsorption to the floor and not to the nearby metal walls. The nanometer size scale and three-dimensional n...

Microfluidic channels with two lateral dimensions smaller than 1 microm were fabricated in fused silica for high-sensitivity single-molecule detection and fluorescence correlation spectroscopy. The effective observation volumes created by these channels are approximately 100 times smaller than observation volumes using conventional confocal optics and thus enable single-fluorophore detection at higher concentrations. Increased signal-to-noise ratios are also attained because the molecules are restricted to diffuse through the central regions of the excitation volume. Depending on the channel geometries, the effective dimensionality of diffusion is reduced, which is taken into account by simple solutions to diffusion models with boundaries. Driven by electrokinetic forces, analytes could be flowed rapidly through the observation volume, drastically increasing the rate of detection events and reducing data acquisition times. The statistical accuracy of single-molecule characterization is improved because all molecules are counted and contribute to the analysis. Velocities as high as 0.1 m/s were reached, corresponding to average molecular residence times in the observation volume as short as 10 micros. Applications of these nanofabricated devices for high-throughput, single-molecule detection in drug screening and genomic analysis are discussed.