Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-07-14
2003-12-09
Nguyen, Nam (Department: 1753)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S450000, C204S451000, C204S600000, C204S601000, C204S605000, C435S288500, C435S288600
Reexamination Certificate
active
06660147
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to electrophoretic analysis of analytes of interest. More particularly, the invention relates to small-scale devices for conducting electrophoretic separation and/or analysis of analytes, as well as chemical and biochemical methods employing such devices.
REFERENCES
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Capillary Electrophoresis: Theory and Practice
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BACKGROUND
The structural analysis of polynucleotides and other biomolecules is playing an increasingly important role in modern molecular biology. With the advent of polynucleotide amplification technology, e.g., PCR, and projects directed towards sequencing the human genome, the level of interest in this area is high. In particular, the need to process large numbers of samples as quickly as possible has led to the need for analytical systems with increased resolution, throughout, and automation.
It would be desirable to have a device which permits efficient, large-scale analysis of many samples in as small an area as possible, in order to reduce cost and the amount of sample manipulation. At the same time, the device should provide reproducible, high sensitivity detection of analytes of interest. Preferably, the device will be compatible with a variety of different sample types and will be amenable to re-use with different sample sets.
SUMMARY
In one aspect, the present invention provides an apparatus for electrophoretic separation of analytes. In a preferred embodiment, the apparatus comprises a planar substrate defining (1) a central reservoir region, (2) a plurality of electrophoretic channels in fluid communication with, and emanating substantially radially from, the central reservoir region, the channels being coplanar with each other, and each channel having (i) a proximal end which is linked to the reservoir region, and (ii) a distal end. At the distal end of each channel, the substrate further defines at least one chamber linked in fluid communication with the distal end of the channel. For example, each channel can be linked to a sample chamber, a sample-receiving chamber, and a running buffer chamber. Alternatively, each channel can be linked to two distal chambers. Each one or more chambers is preferably linked to the distal end of a channel by a passageway that leads from each chamber in a direction that is initially away from the central reservoir region, whereby centrifugation of the substrate about a central axis that is perpendicular to the channels is effective to disperse liquid from the central reservoir region into the channels and chambers, such that any air bubbles in the chambers, channels, and passageways are forced towards the axis of rotation, when such liquid is present in the central reservoir region.
The apparatus preferably includes electrodes for applying a voltage potential between the chambers and the central reservoir. The apparatus may also include a detector for detecting selected components which may be present in the channels. In one embodiment, the detector and substrate are disposed such that the detector and/or substrate are rotatable relative to each other to permit rotary detection. For example, in one approach, the detector can be rotatable about a central axis within the central reservoir region, for detecting signal emission from each of the channels at a selected distance from the axis, or along a selected length of each channel. In an alternate embodiment, the substrate may be rotatable about a central axis such that the channels pass sequentially by the detector, for detecting one or more components that may be present in the channels. In a preferred embodiment, the detector is adapted for detecting a fluorescent or chemiluminescent signal.
In one embodiment, the apparatus may include an annular septum that covers, and which may partially define, the chambers, and which permits needle access to the chambers for delivery of liquids to the chambers.
In another embodiment, one or more of the channels may contain an electrophoresis medium, such as a covalently crosslinked medium, a noncovalently crosslinked medium, or a flowable medium.
In another aspect, the invention provides a method for preparing a plurality of electrophoretic paths which are substantially bubble-free. The method may include providing an apparatus such as described above, such that the reservoir region contains a liquid or is in fluid communication with a liquid source, and centrifuging the substrate about a central axis that is perpendicular to the channels so that the liquid is dispersed from the central reservoir region into the channels and chambers, such that any air bubbles in the chambers, channels, and/or passageways are forced towards the axis of rotation, yielding a plurality of bubble-free electrophoretic paths between the reservoir and the chambers.
In an alternate embodiment, the method may include providing an apparatus such as described above such that the reservoir region, and optionally the channels, passageways, and/or chambers, contain a liquid, and centrifuging the substrate about a central axis that is perpendicular to the channels so that the liquid is dispersed from the central reservoir region into the channels and chambers, such that any air bubbles in the chambers, channels, and/or passageways are forced towards the axis of rotation, yielding a plurality of bubble-free electrophoretic paths between the reservoir and the chambers.
The apparatus and methods discussed above can also be used for sample analysis. In one aspect, the invention includes a method for analyzing a plurality of samples. The method preferably includes providing an apparatus such as describes above, such that the central reservoir region, channels, and chambers contain a liquid medium suitable for electrophoresis of such samples. Samples are provided in one or more of the sample chambers, and an electric field is applied under conditions effective to cause migration of sample(s) through at least one channel towards the central reservoir region. The channel(s) may be interrogated before, during and/or after electrophoresis to detect one or more sample components in the channel(s).
The invention may be applied to the separation and/or analysis of any of a variety of samples, particularly proteins, nucleic acids, polysaccharides, small molecules, and the like. Also, sample components to be detected may be labeled with detectable labels, e.g., fluorescent or chemiluminescent labels, to aid detection. The invention is also useful in combination with a wide variety of sample preparation methods, such as the polymerase chain reaction, oligonucleotide ligation assays, restriction fragment analysis, polymer sequencing, screening assays, and the like.
Th
Kowallis Reid B.
Woudenberg Timothy M.
Applera Corporation
Nguyen Nam
Powers Vincent M.
Starsiak Jr. John S.
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