Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-12-17
2002-08-27
Warden, Jill (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S450000, C204S451000, C204S452000, C204S453000, C204S454000, C204S600000, C204S601000, C204S605000, C435S287200, C422S068100, C427S105000, C427S106000, C427S107000, C436S086000, C436S089000, C210S198200
Reexamination Certificate
active
06440284
ABSTRACT:
BACKGROUND OF THE INVENTION
Capillary electrophoresis has been established as a highly effective method for separating macromolecular species in order that they might be further characterized. Protein and nucleic acid molecules are two major examples of molecular species that are routinely fractionated and characterized using capillary electrophoretic systems. These systems have generally proven effective as a result of the high surface to volume ratio of the thin capillaries. This high surface to volume ratio allows for much greater heat dissipation, which in turn, allows application of greater electrical currents to the capillary thereby resulting in a much more rapid separation of macromolecules introduced into the system.
In the capillary electrophoretic, size-based separation of biological macromolecules of interest, e.g., proteins and nucleic acids, electrophoretic separation is not possible in a free solution. Instead, such separation requires the presence of a matrix that alters the electrophoretic mobilities of these molecules based upon their relative size.
Although early capillary electrophoresis systems utilized solid gel matrices, e.g., cross-linked polyacrylamides, more recent systems have employed liquid polymer solutions as a flowable matrix, which permits adequate separation efficiencies without the drawbacks of cross-linked capillary systems, i.e., in introducing such matrices to or removing them from capillary channels.
For example, U.S. Pat. No. 5,126,021 reports a capillary electrophoresis element which includes a capillary electrophoresis tube containing a low viscosity uncharged polymer solution, for separating nucleic acids.
U.S. Pat. No. 5,264,101 to Demorest et al. reports the use of a hydrophilic polymer solution, which is characterized by a molecular weight of 20 to 5,000 Kd, and a charge between 0.01 and 1% as measured by the molar percent of total monomer subunits to total polymer subunits, where the charge is opposite to the charge of the surface of the capillary in which the polymer is used. This opposite charge of the polymer is reported to result in an interaction between the polymer and the capillary wall to reduce electroosmotic flow within the capillary.
U.S. Pat. Nos. 5,552,028 and 5,567,292, both to Madabhushi et al., report the use of a uncharged, water soluble, silica adsorbing polymer in a capillary electrophoresis system to reduce or eliminate electroosmotic flow.
Surprisingly, the present inventor has discovered that polymer solutions can be used in capillary channel systems, which polymers employ a charge that is the same as that of the internal capillary surface, e.g., positive or negative. Even more surprisingly, it has been discovered that electroosmotic flow in capillary channel systems containing such polymer solutions is maintained the same level or lower than with an uncharged polymer solution. The present invention provides such polymers, as well as methods of utilizing these polymers and systems employing such polymers.
SUMMARY OF THE INVENTION
The present invention generally provides novel methods and compositions for use in the separation of molecular, and particularly macromolecular species by electrophoretic means.
For example, in an aspect of the present invention is provided a method of separating macromolecules by capillary electrophoresis. The method generally comprises providing a substrate which includes at least a first capillary channel disposed therein, where a surface of the channel has a first surface charge associated therewith. The capillary channel is filled with a water soluble hydrophilic polymer solution which includes a percent charge of from about 0.01% to about 2%, as calculated by the molar percent of charged monomer subunits to total monomer utilized in producing the polymer. The charged monomer subunits have a charge that is the same as the first surface charge. A sample containing macromolecules is introduced into one end of the capillary channel and a voltage gradient is applied across the length of the capillary channel, whereby the macromolecules in the sample are separated in the capillary channel. In preferred aspects, the surface charge of the capillary channel, as well as the charged monomer subunits bear a negative charge. In further preferred aspects, the capillary channel is disposed within a silica substrate.
In a related aspect, the present invention also provides systems and apparatus for practicing the above methods. In particular, the present invention provides a system for separating macromolecules by capillary electrophoresis. The system comprises a substrate having at least a first walled capillary channel disposed therein, where the channel includes a net surface charge associated with its interior surfaces. A solution of silica adsorbing polymer as described above, is disposed in the capillary channel. The system also includes a power source electrically coupled to the capillary channel for applying a voltage gradient across the capillary channel.
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Caliper Technologies Corp.
Murphy Matthew B.
Quine Jonathan A.
Starsiak Jr. John S.
Warden Jill
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