Fast high-resolution mapping of long fragments of genomic DNA based on single-molecule detection.

Here we describe bacterial genotyping by direct linear analysis (DLA) single-molecule mapping. DLA involves preparation of restriction digest of genomic DNA labeled with a sequence-specific fluorescent probe and stained nonspecifically with intercalator. These restriction fragments are stretched one by one in a microfluidic device, and the distribution of probes on the fragments is determined by single-molecule measurement of probe fluorescence. Fluorescence of the DNA-bound intercalator provides information on the molecule length. Because the probes recognize short sequences, they encounter multiple cognate sites on 100- to 300-kb-long DNA fragments. The DLA maps are based on underlying DNA sequences of microorganisms; therefore, the maps are unique for each fragment. This allows fragments of similar lengths that cannot be resolved by standard DNA sizing techniques to be readily distinguished. DNA preparation, data collection, and analysis can be carried out in as little as 5h when working with monocultures. We demonstrate the ability to discriminate between two pathogenic Escherichia coli strains, O157:H7 Sakai and uropathogenic 536, and we use DLA mapping to identify microorganisms in mixtures. We also introduce a second color probe to double the information used to distinguish molecules and increase the length range of mapped fragments.

Staphylococcus aureus strain typing by single-molecule DNA mapping in fluidic microchips with fluorescent tags.

BACKGROUND: Epidemiologic studies require identification or typing of microbial strains. Macrorestriction DNA mapping analyzed by pulsed-field gel electrophoresis (PFGE) is considered the current gold standard of genomic typing. This technique, however, is difficult to implement because it is labor-intensive and difficult to automate, it requires a long time to obtain results, and results often vary between laboratories. METHODS: We used direct linear analysis (DLA), which uses a single reagent set and long fragments of microbial genomic DNA to identify various microbes. In this technique, an automated system extracts fragments exceeding 100 kb from restriction enzyme digests of genomic DNA from microbial isolates and hybridizes them with a sequence-dependent fluorescent tag. These fragments are then stretched in a microfluidics chip, and the patterns of the distribution of the tags are discerned with fluorescence confocal microscopy. The tag pattern on each DNA fragment is compared with a database of known microbial DNA sequences or with measured patterns of other microbial DNAs. RESULTS: We used DLA to type 71 Staphylococcus aureus strains. Of these, 9 had been sequenced, 10 were representative of the major pulsed-field types present in the US, and 52 were isolated recently in a hospital in Cambridge, MA. Matching DNA fragments were identified in different samples by a clustering algorithm and were used to quantify the similarities of the strains. CONCLUSIONS: DLA-based strain typing is a powerful technique with a resolution comparable to macrorestriction mapping with PFGE, but DLA is faster, more automated, and more reproducible.

Formation of fibers by electrospinning.

Electrostatic fiber formation, also known as "electrospinning", has emerged in recent years as the popular choice for producing continuous threads, fiber arrays and nonwoven fabrics with fiber diameters below 1 microm for a wide range of materials, from biopolymers to ceramics. It benefits from ease of implementation and generality of use. Here, we review some of the basic aspects of the electrospinning process, as it is widely practiced in academic laboratories. For purposes of organization, the process is decomposed into five operational components: fluid charging, formation of the cone-jet, thinning of the steady jet, onset and growth of jet instabilities that give rise to diameter reduction into the submicron regime, and collection of the fibers into useful forms. Dependence of the jetting phenomenon on operating variables is discussed. Continuum level models of the jet thinning and jet instability are also summarized and put in some context.

Electrospun poly(styrene-block-dimethylsiloxane) block copolymer fibers exhibiting superhydrophobicity.

Block copolymer poly(styrene-b-dimethylsiloxane) fibers with submicrometer diameters in the range 150-400 nm were produced by electrospinning from solution in tetrahydrofuran and dimethylformamide. Contact angle measurements indicate that the nonwoven fibrous mats are superhydrophobic, with a contact angle of 163 degrees and contact angle hysteresis of 15 degrees . The superhydrophobicity is attributed to the combined effects of surface enrichment in siloxane as revealed by X-ray photoelectron spectroscopy and surface roughness of the electrospun mat itself. Additionally, the fibers are shown by transmission electron microscopy to exhibit microphase-separated internal structures. Calorimetric studies confirm the strong segregation between the polystyrene and poly(dimethylsiloxane) blocks.

Controlling the fiber diameter during electrospinning.

We present a simple analytical model for the forces that determine jet diameter during electrospinning as a function of surface tension, flow rate, and electric current in the jet. The model predicts the existence of a terminal jet diameter, beyond which further thinning of the jet due to growth of the whipping instability does not occur. Experimental data for various electrospun fibers attest to the accuracy of the model.

Electrospinning Bombyx mori silk with poly(ethylene oxide).

Electrospinning for the formation of nanoscale diameter fibers has been explored for high-performance filters and biomaterial scaffolds for vascular grafts or wound dressings. Fibers with nanoscale diameters provide benefits due to high surface area. In the present study we explore electrospinning for protein-based biomaterials to fabricate scaffolds and membranes from regenerated silkworm silk, Bombyx mori, solutions. To improve processability of the protein solution, poly(ethylene oxide) (PEO) with molecular weight of 900,000 was blended with the silk fibroin. A variety of compositions of the silk/PEO aqueous blends were successfully electrospun. The morphology of the fibers was characterized using high-resolution scanning electron microscopy. Fiber diameters were uniform and less than 800 nm. The composition was estimated by X-ray photoelectron spectroscopy to characterize silk/PEO surface content. Aqueous-based electrospining of silk and silk/PEO blends provides potentially useful options for the fabrication of biomaterial scaffolds based on this unique fibrous protein.