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  Methods and devices for removal of organic molecules from biological mixtures using a hydrophilic solid support in a hydrophobic matrixUSPTO Application #: 20080103297Title: Methods and devices for removal of organic molecules from biological mixtures using a hydrophilic solid support in a hydrophobic matrix Abstract: Methods and devices for removing small negatively charged molecules from a biological sample mixture that uses a solid-phase extraction material that includes a hydrophilic solid support at least partially embedded within a hydrophobic matrix. (end of abstract) Agent: 3m Innovative Properties Company - St. Paul, MN, US Inventors: Ranjani V. Parthasarathy, Raj Rajagopal, Vicky L. Morris, William Bedingham, Barry W. Robole USPTO Applicaton #: 20080103297 - Class: 536025400 (USPTO) Related Patent Categories: Organic Compounds -- Part Of The Class 532-570 Series, Azo Compounds Containing Formaldehyde Reaction Product As The Coupling Component, Carbohydrates Or Derivatives, Nitrogen Containing, Dna Or Rna Fragments Or Modified Forms Thereof (e.g., Genes, Etc.), Separation Or Purification Of Polynucleotides Or Oligonucleotides The Patent Description & Claims data below is from USPTO Patent Application 20080103297. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10/027,226, filed Dec. 20, 2001, now allowed, the disclosure of which is incorporated by reference in their entirety herein. BACKGROUND [0002] Water-soluble dyes (e.g., fluorescent, chemiluminescent, visible, and near-IR) are used routinely in molecular biology to label and monitor components of biological reactions. Frequently, residual dyes as well as other organic molecules should be removed before proceeding with many downstream applications. Thus, the present invention is directed to removing dyes and other organic molecules from biological mixtures, particularly in low volume, microfluidic devices. [0003] There is a significant need for high throughput, low volume, integrated microfluidic devices in order to increase sample throughput and reduce the amount of reagents used per sample (thereby reducing cost per sample) in biological reactions. Small volume Polymerase Chain Reaction (PCR) and nucleic acid cycle sequencing reactions are examples of standard molecular biology techniques that are suitable for incorporation into miniaturized formats. In both applications, removal of residual primers, nucleic acid templates, dyes, and other organic molecules are generally necessary prior to any further downstream applications. [0004] One example where such removal methods are used is in the preparation of a finished sample (e.g., purified nucleic acid materials) from a starting sample (e.g., a raw sample such as blood, bacterial lysate, etc.). For example, to obtain a purified sample of the desired materials in high concentrations, the starting sample is typically prepared for PCR after which the PCR process is performed to obtain a desired common PCR reaction product. The common PCR reaction product can then be used in a variety of molecular biological applications, including, for example, sequencing, cloning, genotyping, and forensic applications. [0005] In fluorescence-based DNA sequencing applications, unincorporated dye terminators (i.e., dye-labeled dideoxy terminators such as dideoxynucleotide triphosphates (ddNTPs)) should preferably be removed from the reaction mixture prior to analysis of the DNA sequence fragments. Failure to sufficiently reduce the concentration of dye terminator molecules leads to dye artifacts (i.e., other dye-containing molecules such as dye-labeled dideoxy terminators such as dideoxynucleotide diphosphates (ddNDPs), dideoxynucleotide monophosphates (ddNMPs), and dideoxynucleosides) that can significantly obscure DNA sequence information. Sequencing reaction purification is a desired step in the preparation of samples prior to sequence analysis, particularly when using a capillary electrophoresis (CE) sequencer. [0006] Conventionally, after completion of the PCR or cycle sequencing reaction, the product is generally purified by either alcohol (ethanol or isopropanol) precipitation or gel filtration chromatography. Other protocols using polyalkylene glycol and biotin-streptavidin interactions have also been utilized for sequencing reaction clean-up. Ultrafiltration membranes, phenol/chloroform extraction, and enzymatic treatments are other methods that are commonly used for purification of PCR and sequencing reaction mixtures. [0007] Such conventional technologies for the purification of PCR and nucleic acid sequencing reactions have not proven to be suitable for incorporation into a microfluidic device. Alcohol precipitation utilizes volatile and flammable reagents. Hydrogels (e.g., crosslinked dextrans), commonly used in size exclusion chromatography, require large bed volumes (10.times. relative the volume of sample) for efficient separation of impurities from product. Gels are first swollen with a relatively large volume of water, centrifuged, and loaded substantially immediately, because, upon dehydration, these materials are prone to cracking. Biotin-streptavidin mediated purifications require the use of custom biotinylated primers for the efficient capture of product. Biotinylated products are generally captured onto streptavidin-treated paramagnetic particles and physically separated from impurities with the use of a magnet. Alternatively, hybridization based purification (HBP) of the PCR or nucleic acid sequencing product can be accomplished by utilizing primers containing specially designed capture tags. Separation of the nucleic acid fragment from the biological matrix can be achieved by hybridization of the capture tag to a complementary strand bound to a solid support. Both the biotin and HBP strategies would require a rinsing step followed by elution of the sequencing or PCR product from the substrate. Although biotin-streptavidin and HBP purification methods yield clean PCR and sequencing fragments, both approaches require customized primers, which can be cumbersome and expensive. [0008] An alternative approach for the removal of residual dye terminators from DNA sequencing reactions involves treating the reaction mixture with an enzyme (e.g., shrimp alkaline phosphatase) to dephosphorylate residual nucleotide triphosphates. Although cleavage of the phosphate groups(s) from the dye-labeled dideoxynucleotide triphosphates alters the mobility of the dye-labeled nucleotides in the sequencing gel, residual dye moieties are not removed from the reaction mixture by this procedure and must still be eliminated prior to injection of the sample into the sequencer. This is generally accomplished by subsequent alcohol precipitation of the digested product. [0009] PCR and sequencing products can also be effectively purified by adsorption of nucleic acid fragments onto beads and silica gel membranes using chaotropic agents. Impurities (e.g., residual primers, dyes, and salts) can be rinsed from the substrate and the purified product eluted. This multi-step bind/rinse/elute purification scheme may also prove to be cumbersome within the context of a microfluidic device. [0010] Yet another method of removing unwanted materials (e.g., dyes) from cycle sequencing (e.g., Sanger cycling) reaction mixtures involves the use of paramagnetic particles. One example of suitable paramagnetic particles incorporating dye terminator removal materials is available under the trade designation RAPXTRACT from Prolinx Inc., Bothell, Wash. Further examples of these materials (and their methods of use) may be found in U.S. patent application Ser. No. 09/894,810 filed on Jun. 28, 2001 and entitled ENHANCED SAMPLE PROCESSING DEVICES SYSTEMS AND METHODS (U.S. Pat. Application Publication No. 2002/0047003 (Bedingham et al.)). Unfortunately, however, with such particles, the particles must remain in a hydrated state, which limits the ability to prefabricate particle-loaded devices. [0011] Thus, methods are needed for the removal of dyes and other organic molecules from biological mixtures, such as nucleic acid amplification reaction mixtures (e.g., PCR or cycle sequencing reaction mixtures). SUMMARY OF THE INVENTION [0012] The present invention provides a solid-phase extraction material that includes a hydrophilic solid support (typically in the form of particles) at least partially embedded within a hydrophobic matrix. The present invention also provides methods for processing biological mixtures, i.e., samples containing a biological material such as peptide- and/or nucleotide-containing material, using such solid-phase material. Specifically, the present invention provides methods for the removal of negatively charged organic molecules (e.g., dyes, primers, probes, dNTPs, dye terminators such as ddNTPs, ddNDPs, ddNMPs, and nucleosides) from biological sample mixtures using hydrophilic particles at least partially embedded within a hydrophobic matrix. These methods are based on solid-phase extraction techniques. They are advantageous because they can be incorporated into high throughput, low volume, integrated microfluidic devices, if desired, particularly those being developed for PCR and DNA sequencing. [0013] The present invention provides methods for removing small negatively charged organic molecules (i.e., unwanted molecules) from a biological sample mixture. Preferably, the biological sample mixture is a biological sample mixture such as a nucleic acid amplification reaction mixture (e.g., a PCR reaction mixture or a nucleic acid sequencing reaction mixture). [0014] Herein, "removal" of unwanted molecules involves adhering such molecules to the solid-phase material and allowing desirable products to remain in solution. This is in contrast to conventional elution methods that involve adhering the desirable products to the solid-phase material, washing away the unwanted molecules, and eluting the desirable products to remove them from the solid-phase material. [0015] In one embodiment, a method includes: providing a solid-phase extraction material that includes a hydrophilic solid support (preferably, particles) at least partially embedded within a hydrophobic matrix (preferably, an adhesive); providing a biological sample mixture; and contacting the biological sample mixture with the solid-phase extraction material to remove at least a portion of the small negatively charged organic molecules from the biological sample mixture. Preferably, the hydrophilic solid support is in the form of particles pattern coated on a layer, and at least partially embedded therein, of the hydrophobic matrix (preferably, a silicone, polyvinyl butyral, polyolefin, fluorinated polymer, acrylate, epoxy, natural or synthetic rubber, or combinations (e.g., mixtures, copolymers, terpolymers, etc.) thereof. [0016] In another embodiment, a method includes: providing a device that includes at least one process array that includes a solid-phase extraction material, wherein the solid-phase extraction material includes a hydrophilic solid support (preferably, particles) at least partially embedded within a hydrophobic matrix (preferably, an adhesive); providing a biological sample mixture in the at least one process array; and transferring the biological sample mixture within the at least one process array, wherein the biological sample mixture and the solid-phase extraction material remain in contact for a sufficient time to remove at least a portion of the small negatively charged organic molecules from the biological sample mixture. Preferably, the hydrophilic solid support is in the form of particles pattern coated on a layer of the hydrophobic matrix. [0017] In yet another embodiment, a method includes: providing a device that includes at least one process array that includes a solid-phase extraction material, wherein the solid-phase extraction material includes hydrophilic particles disposed on a layer of a hydrophobic matrix and at least partially embedded therein; providing a biological sample mixture in the at least one process array; and transferring the biological sample mixture within the at least one process array, wherein the biological sample mixture and the solid-phase extraction material remain in contact for a sufficient time to remove at least a portion of the small negatively charged organic molecules from the biological sample mixture. Preferably, the hydrophilic particles are pattern coated on a layer of the hydrophobic matrix. [0018] When the biological sample mixture is a sequencing reaction mixture, the small negatively charged molecules are typically selected from the group consisting of dye-labeled terminators, primers, degraded dye molecules, deoxynucleotide triphosphates, and mixtures thereof. Preferably, for such a sample, the method is carried out under conditions effective to remove substantially all the dye-labeled terminators from the biological sample mixture. [0019] When the biological sample mixture is a PCR reaction mixture, the small negatively charged molecules are typically selected from the group consisting of primers, degraded dye molecules, deoxynucleotide triphosphates, and mixtures thereof. Preferably, for such a sample, the method is carried out under conditions effective to remove substantially all the primers from the biological sample mixture. [0020] The present invention also provides devices that can be used to carry out methods of the present invention. Such devices include analytical receptacles, such as microfluidic devices and microtiter plates, for example. [0021] In one embodiment, the present invention provides a device that includes: a plurality of process arrays that include: a plurality of process chambers, each of the process chambers defining a volume for containing a biological sample mixture; and at least one distribution channel connecting the plurality of process chambers; and a solid-phase extraction material within at least one of the process arrays that includes a hydrophilic solid support (preferably, particles) at least partially embedded within a hydrophobic matrix (preferably, adhesive). Preferably, the device further includes a plurality of valves, wherein at least one of the valves is located along the at least one distribution channel. Preferably, the hydrophilic solid support is in the form of particles pattern coated on a layer of the hydrophobic matrix. Continue reading... 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