Fluorescence detection and size measurement of single DNA molecules

Castro, Alonso; Fairfield, Frederic R.; Shera, E.B.

doi:10.1021/ac00055a004

Cited by 115 publications

(82 citation statements)

References 7 publications

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“…Using this technique detection of single fluorescent proteins [14,15] as well as analyses of fluorescent derivatives of amino acids by capillary electrophoresis [16][17][18] have been performed. The hydrodynamic focusing of flowing streams have also been used to detect and distinguish between different sizes of DNAfragments [19], as well as detection and identification of single fluorophores with high efficiencies [20,21]. Combination of the sheath flow, the use of two objectives to collect the fluorescence and small injection volumes, mass detection limits of only six sulforhodamine 101 molecules have been reported [22].…”

Section: Introductionmentioning

confidence: 99%

Electrophoretic separation and confocal laser‐induced fluorescence detection at ultralow concentrations in constricted fused‐silica capillaries

2003

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Laser-induced fluorescence detection of labeled amino acids in a flowing stream at femtomolar (10(-15)M) concentrations was achieved by using a fused-silica capillary flow-cell comprising a constricted thin-walled detection region with inner diameters (IDs) ranging from 2 to 8 microm. The diameter of the constricted region was made to match a diffraction-limited focus of a uniphase transverse electromagnetic mode (TEM(00)) laser beam. Optimization of capillary dimensions and geometries (i.e., curvature, wall thickness, and outer-inner diameter ratio were performed in order to minimize cylindrical lensing of the focused laser beam. The fluorescence was collected in a confocal optical setup using a 1.3 numerical aperture (NA), 100x oil-immersion objective and a single-photon-counting avalanche diode (SPAD). Under conditions of fluid flow, the constriction in the capillary forces all analytes to traverse across the laser probe volume, resulting in a high sampling efficiency. Fluorescein isothiocyanate-labeled glutamate (FITC-Glu) was electrophoretically separated and detected in capillaries having an ID of 2 microm at the constricted region with detection limits of 250 fM (signal-to-noise ratio (S/N) = 3) in the injected solution.

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Section: Introductionmentioning

confidence: 99%

Electrophoretic separation and confocal laser‐induced fluorescence detection at ultralow concentrations in constricted fused‐silica capillaries

2003

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“…This method in principle allows the measurement of extremely long DNA molecules because the signal increases with the length of the molecule. This technique has been used with traditional methods of flow cytometry to measure length distributions of DNA molecules (3,4). Other groups have imaged restriction enzymes digesting extended single DNA molecules for ''optical mapping'' (5,6).…”

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confidence: 99%

A microfabricated device for sizing and sorting DNA molecules

Chou

Spence²,

Scherer³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

281

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We have demonstrated a microfabricated single-molecule DNA sizing device. This device does not depend on mobility to measure molecule size, is 100 times faster than pulsed-field gel electrophoresis, and has a resolution that improves with increasing DNA length. It also requires a million times less sample than pulsed-field gel electrophoresis and has comparable resolution for large molecules. Here we describe the fabrication and use of the single-molecule DNA sizing device for sizing and sorting DNA restriction digests and ladders spanning 2-200 kbp.Many assays in biology require measurement of the length distribution of DNA molecules in a heterogeneous solution. This measurement is commonly done with gel electrophoresis; the molecules are separated by mobility, from which the lengths are inferred. This method is powerful, yet has some drawbacks. For medium to large DNA molecules the resolution is limited to approximately 10%. Gel electrophoresis is time consuming. It generally takes at least an hour to run the gel, not including the setup time to cast the gel. Furthermore, for large molecules the procedure fails. This problem has been alleviated to some extent by the development of pulsed-field gel electrophoresis (1), but running times can be days.With the development of high affinity intercalating DNA stains (2), it has become possible to directly measure the length of single molecules by quantitating fluorescence. The amount of intercalated dye is proportional to the length of the molecule, so measuring the total fluorescent intensity from a single molecule gives a direct measurement of its length. This method in principle allows the measurement of extremely long DNA molecules because the signal increases with the length of the molecule. This technique has been used with traditional methods of flow cytometry to measure length distributions of DNA molecules (3, 4). Other groups have imaged restriction enzymes digesting extended single DNA molecules for ''optical mapping'' (5, 6).We have developed microfabricated devices to size and sort microscopic objects based on measurement of fluorescent properties. The devices have a network of microfluidic channels and are fabricated from a silicone elastomer by using a replica technique (7). Master molds are made from silicon wafers by using standard micromachining techniques. Because the molds can be reused indefinitely, this method of fabrication allows economical mass production of the devices. The devices were patterned as shown in Fig. 1. This fabrication technique is one of a new set of technologies known as soft lithography. Previous work has demonstrated that elastomers can replicate gratings and other test patterns with high (Ϸ50 nm) resolution and fidelity (8, 9). Although some groups have made large (Ϸ30 m) elastomer structures for capillary electrophoresis (10), there is only one other example of a micron scale fluidic network with the elastomer (11). MATERIALS AND METHODSDevice Fabrication. Negative master devices were fabricated in silicon and use...

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“…Our group (9-12) and others (13)(14)(15)(16)(17) have reported DNA fragment analysis by flow cytometric measurements (FCM). In our flow cytometric approach to DNA fragment sizing, restriction fragments are stained with a fluorescent intercalating dye.…”

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confidence: 99%