Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
  • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
  • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
  • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
  • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
  • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0809—Geometry, shape and general structure rectangular shaped
  • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2300/00—Additional constructional details
  • B01L2300/08—Geometry, shape and general structure
  • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
  • B01L2300/087—Multiple sequential chambers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
  • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
  • B01L2400/0421—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic electrophoretic flow
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/04—Moving fluids with specific forces or mechanical means
  • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
  • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L2400/00—Moving or stopping fluids
  • B01L2400/06—Valves, specific forms thereof
  • B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
  • B01L7/00—Heating or cooling apparatus; Heat insulating devices
  • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples

Definitions

• the present teachings relate to a method of isolating nucleic acids from a biological sample containing nucleic acids and other materials.
• nucleic acid isolation and purification methods require multiple steps and utilize affinity binding of nucleic acids to a matrix, typically, a silica-based matrix. Nucleic acids are bound to the matrix and contaminants are washed off. The nucleic acids are then eluted from the matrix by low salt buffers and heat.
• nucleic acid preparation methods require the presence of wash and elution buffer reservoirs and a waste collection chamber, in addition to an affinity capture chamber, it would be difficult to integrate sample preparation and genotyping reactions in a single microfluidic system.
• a simplified method for isolating and purifying nucleic acids would also be desirable.
• a device having a substrate, a fluid processing pathway is disposed in or on the substrate, an inlet disposed at a first end of the fluid processing pathway, an outlet disposed at a second end of the fluid processing pathway, a reaction chamber disposed along the pathway between the first and second end, and a nucleic acid purification chamber disposed along the pathway between the first end and the reaction chamber.
• the reaction chamber can have a pair of forward and reverse polymerase chain reaction primers for together replicating a target nucleic acid sequence.
• a polymerase chain reaction product purification chamber can also be disposed along the fluid processing pathway between the reaction chamber and the second end.
• the nucleic acid purification chamber and the polymerase chain reaction product purification chamber can include purification material.
• the purification material disposed in the nucleic acid purification chamber can comprise a hydrophilic purification material and/or a hydrophobic purification material.
• a second nucleic acid purification chamber can be disposed along the fluid processing pathway between the nucleic acid purification chamber and the reaction chamber.
• first and second nucleic acid purification chambers are provided wherein the first nucleic acid purification chamber comprises a hydrophilic purification material and the second nucleic acid purification chamber comprises a hydrophobic purification material.
• the first nucleic acid purification chamber can comprise a hydrophobic purification material and the second nucleic acid purification chamber can comprise a hydrophilic purification material.
• the fluid processing pathway can comprise a lysing agent disposed in the fluid processing pathway between the first end and the nucleic acid purification chamber.
• a sample comprising whole blood cells for example, can be lysed and purified along the fluid processing pathway.
• the reaction chamber can comprise a magnesium catalyst and/or a polymerase enzyme pre-loaded therein, for example, loaded therein prior to a sample being loaded therein.
• a method for isolating nucleic acids from cellular waste materials whereby a biological sample is directed into an inlet of a fluid processing pathway and cells in the biological sample are lysed in the fluid processing pathway to form a lysate comprising nucleic acids and other material herein referred to as waste or cellular waste materials.
• the lysate is then directed into a nucleic acid purification chamber comprising a nucleic acid purification material disposed therein.
• Nucleic acids that are not captured by the purification material can be directed downstream to a reaction chamber disposed along the fluid processing pathway.
• a protease can be combined with the lysate.
• the lysate can be directed by spinning the substrate.
• the cellular waste material can be captured from the lysate using the nucleic acid purification material, to form purified nucleic acid.
• the purified nucleic acids can be directed away from the nucleic acid purification chamber.
• the purified nucleic acids can be directed by spinning the substrate. In some embodiments, the purified nucleic acids can be directed by opening a valve along the fluid processing pathway adjacent the nucleic acid purification chamber. [0009] According to some embodiments, the purified nucleic acids can be directed from the nucleic acid purification chamber into a reaction chamber pre-loaded with reaction reagents, for example, with polymerase chain reaction reagents.
• the reaction reagents can comprise, for example, buffer components, nucleotides, and a pair of primers comprising a forward primer and a reverse primer for together replicating a target sequence of nucleic acids.
• the reaction reagents can comprise a polymerase enzyme, a polymerase catalyst, or both.
• the nucleic acid purification material can comprise a hydrophilic size- exclusion ion-exchange material.
• the methods can comprise contacting the nucleic acids with hydrophobic size-exclusion particles, for example, before, after, or at the same time that the nucleic acids are contacted with hydrophilic size-exclusion particles.
• the method can comprise pre-loading in the reaction chamber one or more sequencing primers for replicating a target nucleic acid, that is, for replicating an oligonucleotide comprising a particular sequence of nucleic acids.
• FIG. 1 is an illustration of a process for purifying nucleic acids in a biological sample, according to an embodiment of the present teachings.
• FIG. 2 is a top view of a fluid processing pathway for processing a nucleic acid-containing sample in a device according to an embodiment of the present teachings;
• FIG. 3 is an illustration of an initial step of a method according to an embodiment of the present teachings using the fluid processing pathway of FIG. 2, and showing the pathway in a beginning orientation and containing a loaded sample;
• FIG. 4 is a top view of the fluid processing pathway of FIG. 2 and the region of the pathway where sample loading and sealing occurs;
• FIG. 5 is a top view of the fluid processing pathway of FIG. 2 and the region of the pathway where polymerase chain reaction occurs;
• FIG. 6 is a top view of the fluid processing pathway of FIG. 2 and the region of the pathway where PCR purification occurs;
• FIG. 7 is a top view of the fluid processing pathway of FIG. 2 and the region of the pathway where purification and forward and reverse sequencing reactions occur;
• FIG. 8 is a top view of the fluid processing pathway of FIG. 2 and the region where fluid communications are formed to open the sequencing reaction chambers and enable purified PCR product to be directed into two respective sequencing chambers;
• FIG. 9 is a top view of the fluid processing pathway of FIG. 2 and the region of the pathway where outlets from the sequencing reaction chambers are formed and the sequencing reaction (SR) products are purified in respective sequencing reaction product purification chambers;
• FIG. 10 is a top view of the fluid processing pathway of FIG. 2 and the region of the pathway where purified sequencing reaction product from the forward sequencing reaction and from the reverse sequencing reaction are forced into respective product collection wells; and [0022] FIG. 1 1 depicts the results of gel electrophoresis after PCR amplification of mtDNA from Raji cell lysates treated with different combinations of purification beads. [0023] Other various embodiments of the present teachings will be apparent to those skilled in the art from consideration of the specification and practice of the teachings described herein, and the detailed description that follows. It is intended that the specification and examples be considered as exemplary only.
• the present teachings provide a device and method for separation of nucleic acids from other material, for example, from cellular waste materials such as polypeptides, lipids, salts, short nucleic acid sequences, and carbohydrates.
• the nucleic acids and other material can be mixed together, for example, in a biological sample.
• a method whereby cells from a biological sample can be lysed to form a lysate comprising nucleic acids and other material that will be referred to herein as cellular waste materials.
• the cellular waste material can comprise polypeptides, lipids, carbohydrates, and other material, as described herein.
• a cell lysate 30 comprising nucleic acids 32 and cellular waste materials 34 can be mixed with a waste-binding matrix 36, for example, hydrophilic or hydrophobic purification beads, adapted to capture cellular waste materials 34 from lysate 30.
• Waste-binding matrix 36 can comprise hydrophilic size- exclusion ion-exchange particles and hydrophobic size-exclusion, ion-exchange particles.
• the size-exclusion, ion-exchange particles can comprise, for example, an ion-exchange core micro-encapsulated by a porous shell, for example, a porous polymeric shell.
• the shell can be adapted to capture small molecular weight materials and exclude high molecular weight materials.
• the core can be adapted for ion-exchanging reactions.
• the nucleic acids that are not captured but instead are left in solution can then be directed downstream for subsequent processing and/or analysis. No washing or elution steps are required, as the nucleic acids do not become bound by or otherwise incorporated in or with waste binding matrix 36.
• Exemplary size-exclusion, ion-exchange particles that can be used according to various embodiments are described, for example, in U.S. Patent Application Publication No. US2006/0160122, published July 20, 2006, U.S. Patent Application Publication No. US2006/0051583, published March 9, 2006, U.S. Patent Application Publication No. US2005/0196856, published September 8, 2005, U.S. Patent Application Publication No. US2005/0181378, published August 18, 2005, and U.S. Patent Application Publication No. US2004/0018559, published January 29, 2004, all of which are herein incorporated in their entireties by reference.
• an exemplary molecular weight cut-off of the molecules that are captured as opposed to those that are not captured can be any suitable cut-off, for example, 500 atomic units or 1500 atomic units, based on weight average molecular weight.
• the biological sample that can be purified according to the present teachings can comprise animal-derived biological material such as blood, urine, saliva, or the like body fluid, or a material derived from organisms other than animals, for example, derived from plants or microorganisms.
• the biological sample can comprise tissue homogeneates, cells, cell lysates derived from organisms, cell cultures, or partially purified nucleic acids.
• the nucleic acids that can be purified can comprise deoxyribonucleic acids (DNA) or ribonucleic acids (RNA), for example, double-stranded DNA, single-stranded DNA, plasmid DNA, genomic DNA, cDNA, RNA, RNA derived from exogenous parasites such as viruses, bacteria and fungi, endogenous RNA derived from organisms, tRNA, mRNA, rRNA, siRNA, and combinations thereof.
• DNA deoxyribonucleic acids
• RNA ribonucleic acids
• purification with the purification materials described herein can occur in a reaction chamber disposed along a fluid processing pathway in a substrate, it is to be understood that the purification can also be conducted by contacting a cell lysate with the purification material in a reaction chamber that is not disposed along a fluid processing pathway in or on a substrate.
• the purification can occur in a test tube, beaker, or other separate container prior to loading a resultant purified sample into a PCR reaction chamber.
• FIG. 2 is a top view of a fluid processing pathway 38 of a fluid processing assembly 39 for processing a biological sample, according to various embodiments.
• Exemplary fluid processing assemblies sharing some similar features as fluid processing assembly 39 are described in detail, for example, in U.S. Patent Applications Nos. 10/336,706 and 10/336,330, filed January 3, 2003, and in U.S. Patent No. 7,201 ,881 B2, issued April 10, 2007, U.S. Patent No. 7,198,759 B2, issued April 3, 2007, U.S. Patent No. 6,935,617 B2, issued August 30, 2005, and U.S. Patent No. 7,135,147 B2, issued November 14, 2006, all of which are herein incorporated in their entireties by reference.
• a sample can be processed using assembly 39 and the various method steps illustrated sequentially in FIGS. 3-10. As illustrated in FIGS. 3-10 and discussed below, nucleic acid sample preparation and genotyping reactions can be integrated in a single fluid processing assembly.
• exemplary fluid processing pathway 38 comprises an input chamber 40, a fluid communication 42 between input chamber 40 and a first nucleic acid purification chamber 44, a fluid communication 45 between chamber 44 and a second nucleic acid purification chamber 46, a fluid communication 48 between chamber 46 and a reaction chamber 50, a reaction product purification chamber 52, and a fluid communication 51 between chambers 50 and 52. Also shown in FIGS. 2-10, fluid processing pathway 38 comprises a flow splitter 54 in fluid communication with reaction product purification chamber 52, through a fluid communication 53.
• a forward sequencing reaction chamber 58 and a reverse sequencing reaction chamber 59 can be in fluid communication with flow splitter 54 through respective fluid communications that are generated when a first valve 56 and a second valve 57, respectively, are opened.
• sequencing reaction product purification chambers 64 and 65 Downstream of sequencing reaction chambers 58 and 59 are sequencing reaction product purification chambers 64 and 65 which are respectively in fluid communication with chambers 58 and 59 through fluid communications 60 and 62, respectively.
• Downstream of purification chambers 64 and 65 are output chambers 70 and 72, respectively, which are respectively in fluid communication with chambers 64 and 65 through fluid communications 66 and 68, respectively.
• in fluid communication refers to two features that are in communication with one another by a channel, opening, and/or valve, even if the communication comprises a valve in a closed state but provided that the valve can be opened whereby a fluid can be moved from one of the features to the other.
• FIG. 3 is an illustration of an initial step of a method using assembly 39 of
• Pathway 38 is depicted in an initial state and containing a sample 74 loaded in input chamber 40.
• FIG. 4 is a top view of pathway 38 after sample loading and sealing has occurred.
• an adhesive seal 76 has been placed over the open upper end of input chamber 40 after sample 74 has been loaded into input chamber 40.
• FIG. 4 also shows, by way of the unlabeled directional arrow, the direction of movement of the sample from input chamber 40 into first nucleic acid purification chamber 44. Movement of the sample can be facilitated, for example, by any appropriate force, for example, by gravity, centripetal force, capillary action, pressure differential, or the like.
• any appropriate force for example, by gravity, centripetal force, capillary action, pressure differential, or the like.
• movement of fluid through the device can be facilitated by centripetal force, for example, as provided by spinning a substrate in which or on which fluid processing pathway 38 is formed.
• centripetal force for example, as provided by spinning a substrate in which or on which fluid processing pathway 38 is formed.
• Exemplary systems and methods for moving fluid samples through a processing pathway are shown, for example, in U.S. Patent No. 7,198,759 B2, issued April 3, 2007, which is incorporated herein in its entirety by reference.
• the volume of the first nucleic acid purification chamber can be large enough to contain an entire sample that is input into input chamber 40 while also containing purification material.
• the purification material can comprise hydrophilic and/or hydrophobic purification particles or beads.
• the sample can be directed into first nucleic acid chamber 44 but be prevented from passing into second nucleic acid purification chamber 46 by way of incorporating a valve in a closed position along fluid communication 45.
• Any appropriate valve in a closed position can be provided, for example, a heat-meltable valve, a dissolvable valve, or a deformable valve.
• Exemplary deformable valves that can be used include those described, for example, as Zbig valves in U.S. Patent No. 7,198,759 B2.
• valve along fluid communication 45 can be opened and a sample movement force can be generated to direct the sample from nucleic acid purification chamber 44 into second nucleic acid purification chamber 46. Further movement of the purified sample through fluid processing pathway 38 can be prevented by providing an appropriate valve along fluid communication 48, for example, the same type of valve as used along fluid communication 45.
• the residence time or contact time in each of nucleic acid purification chambers 44 and 46 can be the same or different from one another.
• a hydrophilic purification material can be pre-loaded in nucleic acid purification chamber 44 and a hydrophobic purification material can be pre-loaded in nucleic acid purification chamber 46.
• the contact time can be, for example, at least fifteen seconds, at least thirty seconds, at least forty-five seconds, at least one minute, or at least two minutes, according to various embodiments. In some examples, the contact time can be from forty-five seconds to about sixty seconds or from about thirty seconds to about ninety seconds, independently, in each of first and second nucleic acid purification chambers 44, 46.
• FIG. 5 is a top view of pathway 38 and the region of the pathway 38 where a first reaction with the purified sample can occur.
• the first reaction be comprise, for example, a polymerase chain reaction in reaction chamber 50.
• the sample has been purified in both first nucleic acid purification chamber 44 and second nucleic acid purification chamber 46.
• a valve disposed along fluid communication 48 has been opened and the sample has been moved from second nucleic acid purification chamber 46 through fluid communication 48 and into reaction chamber 50.
• the valve disposed along fluid communication 48 can be reclosed, and a closed valve can be provided along fluid communication 51 downstream of reaction chamber 50.
• An exemplary reclosable valve that can be used along fluid communication 48 can comprise a reclosable valve as described, for example, in U.S. Patent No. 6,935,617 B2, which is incorporated herein in its entirety be reference.
• thermal processing or thermocycling of the contents of reaction chamber 50 can be conducted, for example, as is useful to promote polymerase chain reaction between the purified sample in reaction chamber 50 and PCR reagents that had been pre-loaded in reaction chamber 50.
• reaction chamber 50 can be pre-loaded, for example, with magnesium catalyst, forward and reverse PCR primers, polymerase enzyme, nucleotide triphosphates, and like reagents useful for conducting polymerase chain reaction.
• the pre-loaded reagents can comprise reagents that might otherwise be captured upstream of reaction chamber 50 by purification material in either or both of nucleic acid purification chambers 44 and 46.
• Systems and methods for carrying out thermocycling of the contents of reaction chamber 50 are described, for example, in U.S. Patent No. 7,198,759 B2, and in U.S. Patent Application Publication Nos US 2006/0046304 Al (March 2, 2006), and US2006/0239666 Al (October 26, 2006).
• the valve disposed along fluid communication 51 can be opened, for example, by a deforming action, and the PCR product generated by thermocycling the contents of reaction chamber 50 can be moved in the direction shown by the directional arrow into reaction product purification chamber 52.
• the PCR product can be made to reside in reaction product purification chamber 52, and to thus contact purification material disposed therein, for a period of time.
• the period of time can be at least fifteen seconds, at least thirty seconds, at least forty-five seconds, at least sixty seconds, at least two minutes or for a period of time within a range of from about thirty seconds to about ninety seconds, or from about forty-five seconds to about sixty seconds.
• the reaction product purification material can comprise the size-exclusion, ion-exchange particles described herein, for example, those described in U.S. Patent Application Publication No. US2006/0160122, which is incorporated herein in its entirety by reference.
• the PCR product can be prevented from passing downstream of reaction product purification chamber 52 by providing a closed valve along fluid communication
• valve disposed along fluid communication 53 can be opened, as shown in FIG. 7, and the purified PCR product can be moved into flow splitter 54, again, for example, by centripetal force.
• valves along fluid communications 82 and 84 can be respectively opened and the purified PCR product can be made to flow into sequencing reaction chambers 58 and 59, respectively, as shown in FIG. 8.
• the purified PCR product can be moved in the direction shown by the directional arrows by using centripetal force as described herein.
• Movement of the purified PCR product downstream of sequencing reaction chambers 58 and 59 can be prevented by providing closed valves along downstream fluid communications 60 and 62, respectively.
• the valve along fluid communications 82 and 84 can be closed and thermal processing or thermocycling can be conducted on the purified PCR products in sequencing reaction chambers 58 and 59.
• Sequencing reaction chambers 58 and 59 can be pre-loaded with sequencing reaction reagents, for example, that can be dried down after pre-loading and then solubilized by the solution that contains the purified PCR product.
• flow splitter can be pre-loaded with sequencing reaction reagents, for example, that can be dried down after pre-loading and then solubilized by the solution that contains the purified PCR product.
• the valves along fluid communications 60 and 62 can be opened and the sequencing reaction products can be moved through fluid communications 60 and 62 into sequencing reaction product purification chambers 64 and 65, respectively.
• the sequencing reaction products can be purified in sequencing reaction product purification chambers 64 and 65 for a period of time.
• the period of time can be, for example, at least fifteen seconds, at least thirty seconds, at least forty-five seconds, at least sixty seconds, at least two minutes, or within the range of from about thirty seconds to about ninety seconds, or from about forty-five seconds to about sixty seconds.
• the sequencing reaction products can be prevented from moving downstream of sequencing reaction product purification chambers 64 and 65 by providing closed valves along fluid communications 90 and 92 downstream of sequencing reaction product purification chambers 64 and 65, respectively.
• valves along fluid communications 90 and 92 can be opened and the purified sequencing reaction products can be moved into output chambers 70 and 72, as shown in FIG. 10.
• the purified forward sequencing reaction products and the purified reverse sequencing reaction products can subsequently be removed from output chambers 70 and 72 for further processing, for example, further processing involving capillary electrophoresis.
• a DNA sequencing method can be carried out whereby cells are reacted with a lysis buffer containing enzymes to digest proteins and RNA, followed by capture of non-DNA materials using the purification materials described herein, followed by PCR amplification of the purified DNA, followed by purification, followed by one or more sequencing reactions, followed by one or more other purification steps.
• an RNA sequencing method can be carried out whereby cells are reacted with a lysis buffer containing enzymes to digest proteins and DNA.
• the method can comprise following lysis with the capture of non-RNA materials using the purification materials described herein.
• the purified RNA can be subjected to reverse transcription and PCR amplification, followed by purification.
• the product can be subjected to one or more sequencing reactions, followed by one or more other purification steps.
• a qPCR-based genotyping method can be carried out whereby cells are reacted with a lysis buffer containing enzymes to digest proteins and RNA.
• the method can comprise following lysis with the capture of non-DNA materials using the purification materials described herein.
• the purified DNA can be subjected to real-time PCR amplification, followed by readout/analysis.
• a qPCR-based gene expression method can be carried out whereby cells are reacted with a lysis buffer containing enzymes to digest proteins and DNA.
• the method can comprise following lysis with the capture of non- RNA materials using the purification materials described herein.
• the purified RNA can be subjected to real-time PCR amplification, followed by readout/analysis.
• the purified RNA can be subjected to reverse transcription and then real-time PCR amplification, followed by readout/analysis.
• volume ratios of from one part hydrophilic purification material to ten parts hydrophobic material, to ten parts hydrophilic material to one part hydrophobic material can be used, for example, volume ratios of from one part to five to five parts to one, and volume ratios of about one to one, can be used.
• Tissue culture cells were grown and resuspended in a lysis buffer.
• the lysis buffer included a detergent that broke down the cellular structure (dissolved lipids).
• the lysis buffer also included a protease that broke down the proteins into smaller peptides and amino acids resulting in the release of DNA and RNA.
• RNA was also removed from the mix, leaving undegraded DNA.
• Unmodified beads were used to demonstrate that cell components, other than highly charged high-molecular weight molecules like genomic DNA and mRNAs, are adsorbed by beads that bind to hydrophilic and hydrophilic small molecular weight cellular components.
• a mixture of the following three types of beads was used:
• SEPHADEX G-50 traps small molecular weight charged components (nucleotides, small sugars, salts, ions, peptides, amino acids), (available from Sigma-Aldrich Co., St. Louis, Missouri, Cat. No. G50300, CAS No. 9048-71-9); SEPHAROSE 4B - traps larger components, including peptides, carbohydrates, oligonucleotides (available from Sigma-Aldrich Co., Cat. No. 4B200, CAS No. 9012-36-6); and
• SEPHADEX LH 20 traps lipids, steroids, fatty acids, hormones, and vitamins (available from Sigma-Aldrich Co., Cat. No. LH20100).
• Calf serum Cells were removed from the tissue culture dish having a trypsin treatment. Released cells were collected by centrifugation. The cell pellet was resuspended in 5 ml of Phosphate Buffered Saline and re-centrifuged. The cell pellet was resuspended in lysis buffer (at approximately 5000 cells/ul) containing a strong detergent and the pronase protease. The cells were digested at 37°C for 15 min in 100 uL volumes resulting in the dissolution of lipid structures and of proteins. A 50 ul mixture of beads were added to 100 ul of cell lysates and incubated at room temperature with intermittent mixing for 10 minutes. Different combinations of the following types of beads were used:
• OCTYL SEPHAROSE CL-4B (Sigma-Aldrich) - to capture small molecular weight lipids, steroids, fatty acids, hormones, and hydrophobic substances in the lysate;
• SEPHADEX G50 to remove salts and small molecular weight biomolecules.
• PCR amplification was done with mitoSEQr primers (against human mtDNA) using 0.6uM of primers in 2x Fast PCR master mix with 2ul of gDNA. Cycling conditions comprised heating to 95°C for one minute followed by 40 cycles of fast PCR. Each cycle of fast PCR comprised heating the sample at 95°C for five seconds, then heating the sample at 60°C for 20 seconds, followed by heating the sample at 72°C for 25 seconds.
• FIG. 1 1 depicts the results of the gel electrophoresis. As shown in FIG. 11, successful PCR amplification was seen with most combinations of the beads. The combinations differed in the proportion of each type of beads.
• Bead combination A comprised one volume of SEPHADEX G50, one volume of SEPHAROSE 4B, and one volume of OCTYL SEPHAROSE CL-4B, mited together.
• Bead combination B comprised one volume of SEPHADEX G50, six volumes of SEPHAROSE 4B, and three volumes of OCTYL SEPHAROSE CL-4B.
• Bead combination C comprised six volumes of SEPHADEX G50, one volume of SEPHAROSE 4B, and three volumes of OCTYL SEPHAROSE CL-4B.
• Bead combination D comprised seven volumes of SEPHADEX G50, no SEPHAROSE 4B, and three volumes of SEPHAROSE CL-4B.
• Bead combination E comprised no SEPHADEX G50, seven volumes of SEPHAROSE 4B, and three volumes of OCTYL SEPHAROSE CL-4B.
• Bead combination F comprised one volume of SEPHADEX G50, one volume of SEPHAROSE 4B, and no SEPHAROSE CL-4B

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