Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
1999-12-22
2001-09-04
Phasge, Arun S. (Department: 1741)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
C204S640000, C210S638000, C210S645000, C210S321870, C210S321890
Reexamination Certificate
active
06284117
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the lowering of small and charged molecule concentration from a solution. More particularly, this invention relates to the lowering of ion concentration and relatively small molecules from solutions containing biological materials such as nucleic acids, proteins, and cells. Even more specifically, this invention relates to the lowering of ion concentration and removing oligonucleotide primer molecules in samples containing biological materials such as amplified nucleic acids in very small volumes such as used in microarray assay environments.
BACKGROUND OF THE INVENTION
The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to the invention.
There has been much work in the field of desalting solutions in macroscopic environments. For example, devices and methods have been developed for removal of charged particles, such as ions, using various forms of chromatography including dialysis across permeable membranes, ion exchange resins, size exclusion resins, and electrodialysis and the like. Typically, these macroscale devices and methods have involved the use of passive removal or exchange of materials by diffusion. These systems require, generally, large volumes of fluids to accomplish the desired ion removal or exchange and achieve the desired ionic strength.
For example, desalting using permeable membranes generally involves use of dialysis tubing having a pore size cut-off that allows various sized materials to pass through the membrane. Such processes might also use a planar membrane whereby a solution to be desalted is passed next to said membrane and ions are exchanged by diffusion. In such systems, the solution is re-circulated for an extended period of time until the ionic strength of the solution is reduced.
In another example, ion exchange resins are typically employed to desalt a solution wherein the solution is passed directly over the exchange resin, such as in a column. Like dialysis, this requires copious amounts of solution volume. Additionally, such a means of desalting presents very large surface area over which a sample, with its target materials, must pass thereby allowing valuable target materials to be lost from the sample by nonspecific binding. Size exclusion resins are used in a similar fashion and present the same types of problems.
In still another example, electrodialysis has been used as applied to desalting copious volumes of water. Specifically, such systems have been used successfully to desalinate sea water wherein charged permselective membranes trap ions with like charge behind similarly charged membranes in a direct current field. (Spiegler, Salt Water Purification, 2
nd
ed., Plenum Press, New Your, 1977). Systems such as this that use electronic potential have additional drawbacks in that with increasing time of electrolysis, there is an increasing drop in voltage potential across the permselective membrane due to the buildup of charge across the membrane and this causes decrease in desalinization efficiency. This efficiency problem has been addressed by employing ion exchange resins to sequester the ions once they have been transported across the membrane thereby reducing the local ion concentration. (see U.S. Pat. Nos. 5,316,637, 4,632,745, 5,593,563, and 5,026,465)
Major drawbacks to each of the above methods include the rate at which desalting can occur as well as a limitation of the degree to which desalting can occur. Generally, in such systems, desalting cannot be carried out in an economical fashion to the levels necessary for applicability to micro-volume scales, especially those systems which require use of electronic potentials applied to the micro-volume to induce transport of molecules within the volumes from one point to another. With respect to the current invention, such systems comprise electronically addressable microarrays used in the amplification, isolation, and identification of nucleic acids, proteins, and cells.
Given that there is still a need in the arts for devices and methods capable of efficiently and quickly desalting small volume samples used in connection with electronically addressable microarrays, we have solved such problems by providing a device and method capable of desalting a low volume sample generally in less than 15 minutes, usually in less than 5 minutes, and preferably in less than 3 minutes, to a level of ionic strength, generally less than 100 uS/cm, and preferably less than 50 uS/cm, wherein said sample can be applied to an electronically addressable microarray and analyzed.
SUMMARY OF THE INVENTION
Embodiments of the current invention address problems caused by high ionic strength conditions of low volume solutions containing molecules of interest. Particularly, this invention provides apparatus and methods for lowering ion concentration in such solutions to a level that will allow electronic transport of the molecules of interest without interference from free ions or other charged small molecules in the solutions. For example, it has been observed that problems with transport are encountered with nucleic acids following amplification reactions (e.g., PCR and strand displacement amplification (SDA)) wherein the reaction solutions use high ionic strength conditions. Prior to this invention ionic strength could only be lowered by diluting the reaction solution or using a desalting column either of which resulted in loss of target due to reduction in concentration, insufficient reduction of ionic strength, and further loss of time in completion of the assay.
The high ionic strength level in a sample solution inhibits electronic transport of molecules that are sensitive to an electric field, such as nucleic acids. This is because as electronic potential is applied to the solution, if ions are present, they tend to carry charge and are transported instead of the larger molecules of interest. Thus, ions will migrate to electrode pads of an electronically addressable microarray such as those designated the “APEX chip” as disclosed in U.S. Pat. No. 5,632,957 herein incorporated by reference, instead of the larger molecules of interest.
We have found that desalting microsolutions such as that used in connection with electronically addressable microarrays not only requires an efficient means by which the solution can be desalted to appropriate levels of ionic strength, but also requires an efficient and quick method and apparatus for desalting assay solutions for integration into a cartridge format for in-line desalting coupled to the microarray assay.
Thus, in one embodiment of the invention, an apparatus having elements of the invention is integrated onto the cartridge containing the microarray for in-line desalting of the assay solution prior to the solution's introduction to the microarray.
In another embodiment, the apparatus of the invention comprises a tubular molecular weight cut off membrane that generally has a molecular weight cut off no greater than 500 kDa. The invention having a pore size of this limit allows for the easy exchange of most small charged molecules and ions without allowing loss of larger proteins, nucleic acids, and cells of interest. Generally, it is contemplated that the cut off will allow ions to pass but not larger molecules. In other embodiments, the cut off may be set to allow small nucleic acid molecules such as oligo primers to pass.
In a further embodiment, the apparatus comprises lumen diameters of the tubular membrane that are useful in the transport of various sized structures such as molecules versus whole cells.
In a further embodiment, the apparatus may comprise more than one tubular membrane positioned in parallel to one another for processing different samples simultaneously. Where at least one tubular membrane is contemplated, it is contemplated that sampl
Bloch Kenneth A
Jimenez Manuel
Landis Geoffrey C.
Mehta Prashant P.
Sheldon Ed
Lyon & Lyon LLP
Nanogen Inc.
Phasge Arun S,.
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