Patent Application
20140039177
The invention relates to a method for isolating biomolecules from a biological sample, comprising biomolecule extraction, buffer reconstitution and elution. The invention particularly relates to a method used for isolating nucleic acids.
Preparation and manipulation of high quality nucleic acid is a significant step in molecular biology. The purified nucleic acids isolated from various sources are required for subsequent molecular or forensic analysis. Various methods can be used to extract, isolate and purify nucleic acids for a variety of applications, such as analyte detection, sensing, forensic and diagnostic applications, genome sequencing, and the like. The conventional methods for nucleic acid sample preparation generally include isolation of the sample, extraction of the intracellular components, purification of the nucleic acids, and post-processing treatment for stabilizing the end product. However, the conventional method is a time consuming, labor intensive process with a risk of contamination and nucleic acid degradation.
A number of methods and reagents for nucleic acid isolation and purification have been developed to allow the direct coupling of nucleic acids onto solid supports followed by extraction, such as solid phase extraction technology. Solid-phase extraction (SPE) technology has been leveraged to reduce the extraction times of high purity nucleic acids for sequencing and other applications. SPE techniques are typically performed using a siliceous or ion exchange material as the solid phase. Porous filter membrane materials, such as cellulose, can also be used for non-covalent or physical entrapment of nucleic acid. However, the porous filter membrane materials are traditionally relegated to nucleic acid storage applications due to low extraction efficiencies of nucleic acid from the matrix and laborious purification from the embedded lytic and stabilization chemicals.
For applications requiring high throughput, robotic solutions allow the sample or reagent handling in SPE processes to be automated. However, the robots are expensive, space consuming, and difficult to move from one place to another, and therefore, are not suitable for use in the field, and incompatible with other analytical devices for further downstream applications. By translating and miniaturizing the bench-top processes, a microfluidic device can eliminate the need for manual intervention between different steps, minimize the size, weight or reagent and power consumption of the device compared to the current robotic platforms. Although microfluidic technology enables a high-speed, high-throughput nucleic acid sample preparation, isolation of nucleic acids in a microfluidic environment typically requires a myriad of external control equipment, including compressed air sources or high pressure syringe pumps.
A significant degradation of the nucleic acids occurs using the conventional elution methods, such as heating or mechanical stress. For example, heating of a matrix to facilitate elution of bound nucleic acids results in a high number of single strand-breaks or a spontaneous depurination followed by cleavage of phosphodiester linkages in the eluted nucleic acids. In some other examples, mechanical stress is induced to facilitate nucleic acid elution from a matrix, which includes agitation by vortexing the matrix bound nucleic acids, repeated pipetting of the nucleic acids, or crushing of the matrix. An extra precaution is desirable for eluting high molecular weight nucleic acids, for example, nucleic acids having a length of above 10,000 to 20,000 nucleotides, and especially above 100,000 nucleotides, as high molecular weight nucleic acids are prone to degradation by mechanical stress, harsh treatment or manual handling. In other methods, nucleic acids containing abasic sites are sensitive to pH above 7, and are degraded on even short exposure to high pH. Therefore, elution method that minimizes number of steps and manual handling is desirable to maintain integrity of the nucleic acid.
There is a long felt need for a method of isolating nucleic acids using an automated fluidic device under reduced nucleic acid degradation condition, with better nucleic acid storage capacity and with minimum human intervention. The need extends to various applications including basic research, forensic study, disease detection, analytical purposes, and more. Therefore, a method for isolating nucleic acids using an automated field-able fluidic device is desirable that includes cell lysis, nucleic acid extraction, and purification processes with minimal human intervention.
One example of a method of isolating nucleic acids from a biological material, comprises applying the biological material on a substrate; applying a fluid to the biological material applied on the substrate; extracting the nucleic acids from the biological material applied on the substrate; and collecting the extracted nucleic acids in a substantially intact form, wherein the collected nucleic acid has a molecular weight greater than or equal to 20 kb.
Another example of a method of isolating nucleic acids from a biological material, comprises applying the biological material on a substrate comprising one or more cell lysis reagents impregnated therein; applying a fluid to the biological material applied on the substrate; extracting the nucleic acids from the biological material; and collecting the extracted nucleic acids in a substantially intact form, wherein the collected nucleic acid has a molecular weight greater than or equal to 20 kb.
One example of a method of isolating nucleic acids from a biological material, comprises adding the biological material to a device, wherein the device comprises: a solid phase extraction matrix comprising one or more cell lysis reagents impregnated therein; a buffer reconstitution substrate comprising an wash buffer reagent and elution buffer reagent impregnated therein; a self-rupturing component comprising a pressure source embedded therein; wherein the substrate, the a buffer reconstitution substrate and the self-rupturing component are operationally coupled to each other, washing the solid phase extraction matrix with a reconstituted wash buffer; and eluting the nucleic acids from the solid phase extraction matrix using a reconstituted elution buffer, wherein the eluted nucleic acid has a molecular weight greater than or equal to 10 kb.
These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
Isolation and purification of nucleic acids from a wide variety of samples including bacteria, plants, blood, or buccal swabs are simplified in a greater extent using various methods. The methods allow extraction of nucleic acids from the matrix and subsequent storage as needed. The methods may be adapted for various downstream applications.
To more clearly and concisely describe the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following description and the appended claims. Throughout the specification, exemplification of specific terms should be considered as non-limiting examples.
The singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. Approximating language, as used herein throughout the specification and claims, may be applied to modify any quantitative representation that could permissibly vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term such as “about” is not to be limited to the precise value specified. In some instances, the approximating language may correspond to the precision of an instrument for measuring the value. Where necessary, ranges have been supplied, and those ranges are inclusive of all sub-ranges there between.
As used herein, the term “electroosmotic membranes” refers to the membranes which are capable of maintaining electroosmotic flow of a fluid using electroosmosis. Electroosmosis is a motion of a fluid containing charged species relative to a stationary charged medium by an application of an externally applied electric field. Electroosmotic flows are useful in microfluidic systems as the flow enables fluid pumping and control of the flow-rate without using mechanical pumps or valves.
As used herein, the term “positive electroosmotic membrane” refers to a porous membrane with surface properties, such that induced electroosmotic flow occurs in the direction of the applied electric field in deionized water. It is known to those skilled in the art that the magnitude and direction of electroosmotic flow is dependent on the operating parameters, including the type of running liquid or buffer system used.
As used herein, the term “negative electroosmotic membrane” refers to a porous membrane with surface properties, such that induced electroosmotic flow occurs in the direction opposing the applied electric field in deionized water. It is known to those skilled in the art that the magnitude and direction of electroosmotic flow is dependent on the operating parameters, including the type of running liquid or buffer system used.
As used herein, the term “porous material” refers to a material with a plurality of pores, wherein the material is macroporous, microporous, or nanoporous. The porous material may form “porous membrane” and “porous electrodes”. The pores can be macropores, micropores or nanopores. In case of micropores, the average pore size may be, for example, less than about 10 microns, or less than about 5 microns, or less than about one micron. In the case of nanopores, the average pore size may be, for example, about 200 nm to about 10 microns, or about 200 nm to about 5 microns, or about 200 nm to about 3 microns. The porous membranes may be made of inorganic materials such as, silicon, alumina, silicon nitride, or silicon dioxide. The porous electrodes may be made of metals such as, platinum (Pt) or gold (Au), or redox materials, such as metal salts or conductive polymers.
As used herein, the term “interspersed” or “intervening” refers to a position of a membrane or an electrode which is present between two other electrodes or two other membranes respectively. For example, a membrane is interspersed means the membrane is disposed between two different electrodes, wherein the electrodes are oppositely charged. In another example, an electrode is intervened or interspersed means the electrode is disposed between two membranes with opposite surface charge. The term “disposed between” is alternatively used herein as “interspersed” or “intervened”.
As used herein, the term “operatively coupled” or “operationally coupled” refers to a functional interaction between one or more components. For example, various components are operatively coupled to each other in the device, wherein the components are connected by a fluidic flow while the device is in operation.
As used herein, the term “substantially intact” form refers to a form of nucleic acids that maintains an overall structural integrity, may be of about 70-80%. For example, the nucleic acid that retains its structural integrity of about 70-80% after elution from a matrix may be referred to as being in a substantially intact form. The device enables purifying substantially intact nucleic acids unlike some of the elution methods from a binding matrix using heat treatment or mechanical stress, as described in background section. The “substantially intact form” means nucleic acids with reduced physical or chemical changes, such as minimal degradation, strand breakage, or chemical-modification of the structural units. The substantially intact form of the nucleic acids are useful for various downstream applications, such as whole genome sequencing, disease detection, identification of mutants, and amplification of nucleic acids. For example, purified human DNA having a length of greater than 20,000 nucleotides is very useful for genome sequencing or disease detection. The purification of a substantially intact form of the nucleic acids is also shown in
As used herein, the term “reduced-degradation condition”, refers to a process of reducing degradation of the nucleic acids while isolating from a matrix without using any harsh condition or treatment on the nucleic acids. The harsh treatment may lead to degradation or fragmentation of the nucleic acids. The harsh condition or treatment may include but is not limited to boiling of the nucleic acids, heating of the nucleic acids at a higher temperature, treating the nucleic acids with a strong detergent or chaotrope or the like. In one embodiment, the elution process adopted by the present method employed an electroosmotic pump or EOP, which exerts fluidic pressure on the nucleic acids attached to the matrix. The use of EOP is an example of a reduced degradation condition.
One or more embodiments of a method of isolating nucleic acids from a biological material, comprise applying the biological material on a substrate comprising one or more cell lysis reagents impregnated therein; applying a fluid to the biological material applied on the substrate; extracting the nucleic acids from the biological material applied on the substrate, and collecting the extracted nucleic acids in a substantially intact form, wherein the collected nucleic acid has a molecular weight greater than or equal to 20 kb.
In some examples of the method, the biological material on a substrate is applied using any external device such as a pipette, a dispenser, or may be through a conduit which is interfaced with or coupled to another device or system. In some aspects, the sample is disposed on the substrate manually using an external device. In some embodiments, the sample may be disposed using a robotic instrument, specifically for application of larger sample volume. In one or more embodiments, the sample may be disposed by a device automatically, wherein the device is pre-programmed for sample application. The automatic disposition of sample reduces the human intervention as well as the probability of contamination or degradation of nucleic acids. The automatic disposition enables retrieving substantially intact nucleic acids from the biological sample.
In one or more embodiments, after applying the biological sample comprising live cells to the substrate, the cells are lysed by impregnated cell-lysis reagents. In some embodiments, the method further comprises hydrating the cell lysis reagent on the substrate to extract the nucleic acids from the biological material. The extracted nucleic acids from the cells are immobilized on the substrate. In some embodiments, the method further comprises immobilizing the nucleic acids on the substrate.
In one or more embodiments, the fluid is applied under high pressure or with high flow rate to wash and/or elute the nucleic acids from the substrate. The extracted nucleic acids are then collected in a substantially intact form, wherein the collected nucleic acid has a molecular weight greater than or equal to 20 kb.
The reduced degradation condition enables isolating high molecular weight nucleic acids, which is desirable for various applications. In one or more embodiments, the elution process of nucleic acid is carried out under reduced-degradation condition. The high molecular weight nucleic acids, such as nucleic acids having molecular weight greater than 10 kb, in a substantially intact form are desirable from the sample. The nucleic acids are extracted and purified by a process without using any harsh treatment that reduces the degradation of the nucleic acids. In some embodiments, the method enables purifying nucleic acids having molecular weight greater than or equal to 20 kb. For example, the mouse genomic DNA having a molecular weight of 20 kb is isolated using the MFM device. In some embodiments, the isolated nucleic acids are greater than 30 kb, as needed, for example when isolating human genomic DNA.
In some examples of the methods, a fluid is applied to the biological material disposed on a substrate at a flow rate of less than or equal to 0.1 ml/volt/cm2/minute to extract nucleic acids from the biological material, and collecting the extracted biomolecules in a substantially intact form.
In some embodiments, the method of isolating nucleic acids from a biological material comprises applying a fluid to the biological material disposed on a substrate at a pressure of greater than or equal to 1 PSI, using a voltage of less than or equal to 25 volts. In some other embodiments, the method comprises applying a voltage of less than or equal to 3 volts, wherein a pressure of greater than or equal to 1 PSI is generated.
One or more embodiments of the method may be performed automatically. In some other embodiments, the method is partially automated. Accordingly, the methods enable extracting and collecting the nucleic acids under minimal human intervention, wherein the nucleic acids comprise deoxyribo nucleic acids, ribonucleic acids and combination thereof.
In one or more examples, the application of fluid is controlled by a controller, and the entire application process is automated. In some embodiments, the device may be a pump coupled to a fluidic circuit through which the fluid flows and is applied to the sample. In some examples of the method, the pump may be a syringe pump, a peristaltic pump or an electroosmotic pump (EOP).
In some examples, the fluid is applied to the biological material at a pressure of greater than or equal to 1 PSI using an EOP. In some embodiments, the pressure of greater than or equal to 1 PSI is generated, using an EOP by applying a voltage of less than or equal to 3 volts. In some embodiments, the fluid applied to the sample may be a wash buffer or an elution buffer, and may be applied sequentially for washing and then elution of the nucleic acids on the substrate.
As noted, the cell lysis reagents lyse the cells to extract nucleic acids. In one or more embodiments, the extracted nucleic acids are washed with applied fluid. In one or more embodiments, the fluid is applied to the substrate for washing the substrate to remove the cell debris, protein, fat or other impurities or unwanted materials present on the substrate. The fluid may also be applied to remove excess reagents present on the substrate. In one or more embodiments, the method further comprises washing the nucleic acids by applying a wash buffer on the substrate. The washing procedure may be repeated for two to five times, or more depending on various requirements, for example, the purity of nucleic acids required or the quality of sample applied.
In other embodiments, the method may comprise eluting the nucleic acids by applying an elution buffer to the biological material on the substrate, followed by collecting the nucleic acids for downstream applications. The pressure of greater than or equal to 1 PSI is generated using a pressure source. In one embodiment of the method, the fluid flow for washing or eluting the nucleic acids are controlled valves, wherein the actuation of the valves is controlled by EOP. The nucleic acids are eluted by pumping. In one embodiment, the nucleic acids are eluted using electroosmotic pumping.
In some embodiments, the nucleic acids isolated from biological material include deoxyribonucleic acids (DNAs) or ribonucleic acids (RNAs). In one embodiment, the nucleic acid is deoxyribonucleic acids (DNAs). In one or more embodiments, the DNA may be genomic DNA, chromosomal DNA, bacterial DNA, plasmid DNA, plant DNA, synthetic DNA, recombinant DNA, amplified DNA and combinations thereof.
In some examples of the methods, the substrate comprises a solid phase extraction matrix, a filtration matrix, a membrane or combinations thereof. In one or more examples, the substrate comprises glass, silica, quartz, polymer and combinations thereof. In one embodiment, the substrate comprises quartz.
In some embodiments, the substrate comprises a microporous substrate, a nanoporous substrate or a combination of both. In various examples of a method, wherein the substrate is a hybrid of a microporous substrate and nanoporous substrate, the method comprises, applying a biological material to the microporous substrate, entrapping nucleic acids extracted from the lysed cells on the microporous substrate, utilizing the microporous substrate as a low pressure lateral flow matrix capable of generating capillary fluid flow to remove cell debris and other impurities. The method further comprises applying an electric potential across the nanoporous substrate to provide a high pressure electroosmotic flow capable of eluting a high molecular weight nucleic acids from the microporous extraction matrix via transverse electro-kinetic flow. In some embodiments, the nanoporous substrates are electroosmotic membranes, and generate electroosmotic flow while applying an electric potential across the membrane, and referred to herein as EOP. The method in this example uses differential movement of biomolecules in the lateral versus transverse direction to obtain substantially pure and intact nucleic acids.
In one or more embodiments, the substrate may have a structural modification, wherein it is physically or chemically modified for adherence or attachment of the nucleic acids. In some examples, the substrate may comprise one or more different surface topographies, which enable better attachment of the nucleic acids. In some embodiments, one or more reagents may be impregnated on the substrate, wherein the reagents include but are not limited to cell lysis reagents, nucleic acid stabilizing reagents, buffer reagents, nucleic acid immobilizing reagents and combinations thereof. In one embodiment, the solid phase extraction matrix further comprises FTA® reagents.
In some embodiments, the substrate comprises a solid phase extraction matrix, a filtration matrix, an isolation matrix or combinations thereof. The term “substrate” is interchangeably used herein as “matrix” or “extraction matrix”. As noted, the device may comprise a solid phase extraction matrix. A substrate, wherein the solid phase extraction method can be performed, is referred to herein as a solid phase extraction matrix. Solid phase extraction is a method that uses a solid phase and a liquid phase to isolate one or more molecules of the same type, or different types, from a material. The solid phase extraction matrix is usually used to purify a sample, in some examples, before using the sample in a chromatographic or other analytical method. The general procedure is to load a material onto the solid phase extraction matrix, wash away undesired components, and then elute the desired molecules with a solvent.
In some embodiments, the substrate may comprise glass, silica, quartz, polymer and combinations thereof. In one embodiment, the substrate, such as a solid phase extraction matrix comprises a siliceous material that is impregnated with the reagents. In one embodiment, the substrate is made of quartz. The density of silanol groups on quartz matrix, when compared to standard silica matrix, may facilitate a faster and easier extraction of the nucleic acids from the biological materials. When compared with a glass based matrix using multiple chaotrope and/or detergent combinations, a quartz-based matrix ensures a higher yield of nucleic acids extracted therefrom, under the same conditions. For example, a quartz solid phase extraction matrix using a potassium iodide (KI) chaotrope yields about 70% nucleic acids when compared to a yield of about 50% using a glass fiber in an Illustra® column, as shown in
In some embodiments, the substrate comprises one or more reagents impregnated therein. The impregnated reagents may comprise a lytic reagent, nucleic acid stabilizing reagent, nucleic acid storage chemical and a combination thereof. In one embodiment, the substrate is a solid phase extraction matrix impregnated with one or more reagents for stabilizing biomolecules. In some embodiments, the solid phase extraction matrix is impregnated with one or more lysis reagents.
In some embodiments, the reagents are impregnated in the substrate, in a dried, semi-dried or wet form. In one or more embodiments, the dried reagents are hydrated with a buffer or a sample for cell lysis. For example, the quartz-based-FTA substrate comprises lysis reagents in the dried form and is hydrated by the sample or buffer to reconstitute the reagents. The quartz-based matrix may be impregnated with stabilizing reagents, wherein the lysis reagent is added separately to the quartz matrix. The reagent may be added to the matrix along with the sample before or after adding the sample.
In one or more embodiments, the lysis reagents may comprise a detergent or a chaotropic agent, weak base, anionic surfactant, chelating agent or uric acid. The detergent is a useful agent for isolating nucleic acids because the detergent has the capacity to disrupt cell membranes and denature proteins by breaking protein: protein interactions. The detergent may comprise an ionic detergent, a non-ionic detergent, or a zwitterionic detergent. The ionic detergent may comprise cationic detergent such as, sodium dodecylsulphate (SDS) or anionic detergent, such as ethyl trimethyl ammonium bromide. Non-limiting examples of non-ionic detergent for cell lysis includes TritonX-100, NP-40, Brij 35, Tween 20, Octyl glucoside, Octyl thioglucoside or digitonin. Some of the zwitterionic detergents may comprise 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO). Some of the detergents are denaturing or non-denaturing in nature. Non-denaturing nonionic detergents may include Triton X-100, bile salts, such as cholate. The zwitterionic detergents may include CHAPS.
In one or more embodiments, the substrate may comprise a chaotrope for lysing cells. Generally, chaotropes break inter and intra molecular non-covalent interactions. Examples of chaotropes include, but are not limited to, potassium iodide (KI), guanidium hydrochloride, guanidium thiocyanate, or urea. The chaotropes may be categorized as weaker chaotropes and stronger chaotropes depending on their strength of denaturation. The weaker chaotropes may be used for lysing cells, without affecting the nucleic acids. The weaker binding chaotropes or their surface chemistry may be beneficial in the electroosmotic flow-based MFM.
In one or more embodiments, the lysis reagent used herein is an FTA®-lysis reagent, interchangeably used herein as FTA® reagents. The FTA reagents may comprise Tris, EDTA and SDS. In a typical procedure, the cells are spotted onto the matrix, wherein the impregnated SDS lyses the cells, and the EDTA inhibits nuclease to stabilize the nucleic acids. The wash with Tris-EDTA (TE) buffer solution removes most of the SDS, and ethanol and/or isopropanol washes removes impurities before elution of the nucleic acids. Such FTA® reagents comprising 50 ul of 2% SDS, 10 mM EDTA, 60 mM Tris solution, are used for cell lysis and nucleic acid purification, and are described in U.S. Pat. No. 5,496,562 entitled “Solid Medium and Method for DNA Storage”.
The biomolecules, such as nucleic acids, are extracted from cells after cell lysis, when the cells are in contact with the matrix-bound lysis reagents. In one or more embodiments, the solid phase extraction matrix is configured to immobilize the nucleic acids after extraction from cells. Typically, nucleic acids are bound to a solid phase extraction matrix by a salt bridge, hydrogen bonding, ionic interaction or physical entanglement. Unlike a cellulose-based matrix, where nucleic acids are physically entangled, the extracted nucleic acids are bound to the glass or quartz-based solid phase extraction matrix using a salt bridge interaction or hydrogen bonding. In one example, the nucleic acids are physically entangled to the FTA®-cellulose membrane, and the release of the nucleic acids were difficult from FTA®-cellulose compared to release from other matrices. In some embodiments, the nucleic acids bind to the glass or quartz-based matrices using salt bridge or hydrogen bonding interactions, whereby, the nucleic acid detachment from those matrices is much easier when compared to some other matrices, such as cellulose. The easy release of nucleic acids from the glass or quartz-based matrix also helps to preclude the need for a harsh treatment on the nucleic acids, such as heating the matrices at high temperatures to elute nucleic acids, which would otherwise increase the degradation of the nucleic acids.
To procure non-degraded nucleic acids, one or more stabilizing reagents may be used. In one or more embodiments, the matrix further comprises one or more stabilizing reagents for storing nucleic acids, which helps in stabilization of the nucleic acids and protect from further degradation. For example, use of chelating agents, such as EDTA serves the purpose of stabilization of the nucleic acids. In some examples, typically EDTA inhibits nuclease, which is an enzyme that degrades nucleic acids. The nuclease inhibition further reduces the rate of degradation of the nucleic acids present on the matrix. The function of the chelating agent is to bind the divalent metal ions, such as magnesium, calcium, or transition metal ions, such as iron. Some of the divalent metal ions, for example, calcium and magnesium are known to promote nucleic acid degradation by acting as co-factors for enzymes, like exonucleases. In addition, the redox reaction of the divalent metal ions such as iron, may also damage nucleic acids by generating free radicals.
In one or more embodiments, the substrate is integrated with a device comprising a reagent-storage location and a self-rupturing component comprising a fluid and a pressure source embedded therein, wherein the substrate, the reagent-storage location and the self-rupturing component are operationally coupled to each other. The device, in one or more embodiments, may be referred to herein as a multifunctional membrane device or MFM device. Unlike conventional paper or membrane-based nucleic acid extraction device, the MFM devices enable purification of nucleic acids under reduced degradation conditions. In other embodiments, a method of isolating nucleic acids from a biological material comprises adding the biological sample to a first layer of the MFM device, washing the first layer (substrate) with the wash buffer; and eluting the nucleic acids from the first layer using the elution buffer.
As noted, one example of method for isolating nucleic acids comprises various steps including sample loading, washing, or eluting the nucleic acids. Referring to
Referring to
Referring to
As noted, the substrate may be integrated with a device comprising at least one buffer reconstitution substrate comprising a reagent-storage location. In some embodiments, the buffer reconstitution substrate comprises a wash buffer storage location and one elution buffer storage location. In some embodiments, one or more valves may be operationally coupled to the wash buffer storage location and the elution buffer storage location. In some embodiments, the valve is also operationally coupled to the substrate, such as a solid phase extraction matrix. In some embodiments, the method further comprises reconstituting one or more buffers in the reagent-storage location, using the liquid stored in the self-rupturing component. The fluid (liquid) stored in the self-rupturing component, may flow to the reagent-storage location for reconstituting the buffers, for example, the wash buffer or elution buffer. The reconstituted buffer then flows through the substrate for washing or elution.
The self-rupturing component comprises at least one pressure source, such as an EOP, wherein the EOP is operationally coupled to the buffer reconstitution substrate. The higher flow rate is obtainable using the EOP, wherein the EOP enables intake of larger sample volumes. In some embodiments, the EOP pulls the sample with high pressure, which enables the device to have a higher sample load volume.
The EOP may be configured to maintain high electric field strength across large pump surface areas, to produce a high pressure output at low running voltages, and only requires a small footprint. In some embodiments, a voltage of about 1 to 25 volts is sufficient to generate the high pressure required for driving the fluidic circuit of the device. In some embodiments, the EOPs comprise a plurality of membranes and electrodes, which solve various problems including, bubble formation or reduced field strength and generate a high pressure even at a lower applied voltage using a simple fabrication technique. Accurately controlled electrode-spacing within a thick and dense network of pores in the EOPs provides a solution for maintaining high electric field strength at low running voltages.
One or more embodiments of the EOP used for nucleic acid extraction, comprise a plurality of membranes comprising one or more positive electroosmotic membranes and one or more negative electroosmotic membranes, a plurality of electrodes comprising cathodes and anodes, and a power source. Each of the positive electroosmotic membranes and negative electroosmotic membranes are disposed alternatively, and wherein at least one of the cathodes is disposed on one side of one of the membranes and at least one of the anodes is disposed on the other side of the membrane and wherein at least one of the cathodes or anodes is disposed between a positive electroosmotic membrane and negative electroosmotic membrane. In one embodiment, the EOP is configured to generate pressure at an applied potential of less than or equal to about 25 V for operating fluids in the device. In one embodiment, the EOP generates a flow rate of about 100 μL/min. Such EOP is structured and fabricated as described in U.S. patent application Ser. No. 13/326,653, entitled “Electroosmotic Pump and Method of Use Thereof”, filed Dec. 15, 2011.
The EOP is a self-contained pump comprising pre-charged electrodes, chargeable electrodes, rechargable electrodes and combinations thereof. In one or more embodiments, the nucleic acids are extracted using an EOP, wherein the EOP is a self-contained EOP. The term “self-contained EOP” refers to an EOP which is devoid of any external power source. The EOPs, as described herein, comprise a plurality of membranes and pre-charged, chargeable or rechargeable electrodes, which eliminate the need for external power sources to drive the EOPs and generate a high pressure even at a lower applied voltage. In one embodiment, the EOP is configured to generate pressure at a chemical potential of about 3 V for operating the device. The EOP comprises a plurality of electrodes comprising a material capable of discharging for about 1 hour while running the pump with a flow rate of about 0.5 μL/min. The use of self-contained high pressure EOPs further reduce the expense and spatial requirements for implementing EOP based fluid control in larger systems and devices. Such EOP is structured and fabricated as described in U.S. patent application Ser. No. 13/429,471, entitled “Self-contained Electroosmotic Pump and Method of Use Thereof”, filed Mar. 26, 2012.
In some embodiments, the EOP may be pre-programmed so that the EOP triggers a first cycle of operation to reconstitute wash buffer and transfer the buffer to the extraction matrix for washing the matrix bound biomolecules. In this embodiment, the EOP may also be programmed, so that after the washing step, the EOP triggers the next cycle to elute the nucleic acids from the matrix. The EOP may be programmed so that each of the cycles (wash or elution) is time-controlled.
In some embodiments, the nucleic acids are extracted using a device comprising two EOPs, wherein one EOP is operationally coupled to the wash buffer reservoir and one EOP is operationally coupled to the elution buffer reservoir. Each of the EOPs operates separately for the washing and elution steps, as shown in
The embodiments, where one EOP drives two different steps, such as washing and elution, the third layer and the second layer are connected through a common conduit, which may have two openings, wherein one is in the wash buffer reservoir as a wash buffer inlet, and another is in the elution buffer reservoir as an elution buffer inlet, as shown in
In one or more examples, the application of fluid for washing or elution may be controlled using one or more valves. In one or more embodiments, the device further comprises at least one valve. In some examples, the valve is disposed between the solid phase extraction matrix and the buffer reconstitution substrate, wherein the valve is operationally coupled to the wash buffer reservoir and the elution buffer reservoir (
In one or more examples of the method may use more than one valve, to control the fluid flow from the wash buffer reservoir to the solid phase extraction matrix and from the elution buffer reservoir to the solid phase extraction matrix. In some embodiments, the valve controls the flow of reconstituted buffer solution to the substrate. In some other embodiments, the valve also prevents the back-flow of reconstituted buffer into the EOP. In the case of back-flow, the reconstituted buffer solution may enter the EOP and change the EOP function by altering the zeta-potentials of the membrane employed in EOP.
The actuation of valves may be used to control the fluid flow, wash cycle and elution cycle through the device to isolate nucleic acids from the biological materials. One or more examples of a method of actuating a valve comprises, operatively coupling the valve with an EOP, flowing a fluid through the EOP, and generating a fluidic pressure to actuate the valve.
The method may further comprise actuating one or more valves to control various parameters during operation. In some embodiments, the valves control the flow rate of the fluid applied to the substrate. In some other embodiments, the valves may control the fluid pressure, while applied to the substrate. In some embodiments, the flow of liquid to the wash buffer storage location and the elution buffer storage location is controlled by one or more valves. At least one of the valves is operationally coupled to the self-rupturing component and the reagent storage location. The valve is operationally coupled to the reagent storage location and the substrate. In this embodiment, the flow of liquid to the buffer reconstitution substrate, comprising wash buffer and the elution buffer, is controlled by one or more valves. The liquid passes from the EOP to the buffer reconstitution substrate for reconstituting the wash buffer reagents or elution buffer reagents.
In one or more examples of methods, the steps of nucleic acid extraction are controlled using one or more controllers. One or more examples of the method further comprises controlling the EOP operation, fluid flow rate, fluid pressure, valve actuation, temperature of the device and fluid circuit, and combinations thereof. The EOP may also be operationally controlled by a controller. In some embodiments, a switch or a controller triggers the washing step and the elution step, as needed. In one embodiment, the controller controls the flow of a fluid through the solid phase extraction matrix, buffer reconstitution substrate and EOP. In one or more embodiments, the controller may be a microcontroller. In one or more embodiments, the device may comprise a control circuit to maintain a constant current or voltage for the EOP, and therefore maintains a constant fluid flow or pressure output during the operation of the device. As noted, in one embodiment, the controller for fluid flow may contain a check valve. In one embodiment, a controller may control the fluid flow by controlling the back pressure, which is generated by the EOP. In this embodiment, the controller is a pressure controller, which controls the EOP to generate a pressure. In one embodiment, the EOP is operationally controlled by a controller for washing and eluting the nucleic acids as per user requirement. In one embodiment, the device comprises a controller to maintain a constant fluid flow by regulating input voltage to the EOP. In some embodiments, the valve itself functions as a controller, while controlling the fluid flow. In one embodiment, the controller controls the overall MFM device to operate, wherein the controller is a switch for operating the device when the device is automated. The controller may be further pre-programmed before the operation depending on the application requirement or user requirement. The controller may comprise a micro controller circuit, wherein the controller may be a digital controller.
In one or more embodiments, the nucleic acids are purified using a device that is structured in multiple layers. In some embodiments, the device comprises a first layer comprising a solid phase extraction matrix, a second layer comprising at least a buffer reconstitution substrate comprising at least one wash buffer storage location and one elution buffer storage location comprising an wash buffer reagent and elution buffer reagent embedded therein respectively, and a third layer comprising at least one EOP, wherein the EOP is operationally coupled to the wash buffer storage location and the elution buffer storage location. The first, second and third layers are operationally coupled to each other, as shown in
The first, second and third layers may be operationally coupled to each other, wherein a fluid flows through the EOP of the third layer to the buffer reservoirs of the second layer. In this way, the first, second and third layers are coupled to each other, when the device is in operation. The first, second and third layers are disposed one after another and may be packaged and integrated together. In some examples, one or more intervening layers may exist between the first, second and third layers of the MFM device.
In one embodiment, the device comprises two valves and two pressure sources, such as EOPs, as shown in
In one or more embodiments, the first layer of the MFM comprises a siliceous membrane that is impregnated with the lytic reagents and storage chemicals typically associated with FTA® products. In some embodiments, the quartz-based “FTA” uses the FTA® reagents from GE with a unique combination of quartz matrix (QMA). The glass or quartz-based FTA lyses and deactivates a wide variety of the bacteria and viruses. The glass or quartz FTA version provides a simple user interface associated with the additional benefit of having a surface capable of chaotrope-driven solid phase extraction. Unlike the cellulose-based FTA® product that requires over two hours drying time for bacterial inactivation, the glass, quartz or silica-based membranes have shown E. coli inactivation within 10 minutes. In some other embodiments, the FTA® reagents impregnated in the quartz matrix are useful for a setting in a field-able device. The quartz-FTA matrix is also more efficient in nucleic acid elution when compared to similar glass or silica-based matrices.
In addition, unlike a typical FTA® paper card, integration of the glass-FTA (GFA, from GE) or quartz-FTA (QMA, from GE) with the EOP within the MFM cartridge enables loading of larger sample volumes, due to chemical interaction of nucleic acid with the card. In addition, the EOP provides an internal or self-contained pressure source for washing and eluting the nucleic acids. In some embodiments, a larger sample size may be accommodated using the quartz-based matrix by continuously pulling nucleic acids through the matrix, eliminating the drying step typically required in FTA processing, followed by elution using an EOP. For example, a larger blood sample, such as a 70 μl sample, may be dried onto the quartz-based FTA matrix, wherein the FTA® reagent can lyse the cells and elute the nucleic acids using the EOP. In some other embodiments, by increasing the number of the solid phase extraction matrices, the load volume or capacity of the sample may be increased. For example, by using three membranes, the load capacity of the device is increased (
In one or more embodiments, the dried buffer salts, lysis reagents or stabilizing reagents are used. As noted, the method employs a device comprising one or more dried reagents in the reagent storage location. As noted, the device comprises a self-rupturing component, such as a hermetically sealed liquid filled reservoir, wherein the hermetically-sealed reservoir is operationally coupled to the reagent storage location chambers. The device may comprise a buffer reconstitution substrate, wherein the dried, semi-dried or wet buffer is reconstituted using a fluid contained inside the self-rupturing component. In some other embodiments, a buffer reconstitution substrate comprising a dried, semidried or wet buffer and the buffer, which is reconstituted using a liquid supplied from outside of the device. In some embodiments, the buffer reconstitution substrate comprises one or more reagents, which can be reconstituted to a wash buffer solution and an elution buffer solution. In some embodiments, air or a second liquid phase may be used to separate multiple, operationally-coupled, liquid chambers to allow a liquid buffer exchange.
The substrate is part of a device, and various components of the device may be assembled as illustrated in
The buffer reconstitution substrate is used for housing the wash buffer and elution buffer, is typically made of a thin substrate, to provide a compact portable nucleic acid purification device requiring minimum pressure for fluid flow through the substrate. Moreover, the thin substrate facilitates faster reconstitution of reagents to the fluid to form the wash or elution buffer during operation. In one or more embodiments, the buffer is reconstituted on a buffer reconstitution substrate.
The composition of the buffer reconstitution substrate may vary. In some embodiments, the buffer reconstitution substrate comprises a metal, polymer, glass, silica or combinations thereof. The buffer reconstitution substrate may be a metallic sheet or bar and the buffer reservoirs are embedded therein. In one or more embodiments, the buffer reconstitution substrate may be a polymeric substrate, such as a cellulose membrane, paper, or nylon matrix. The polymeric buffer reconstitution substrate may comprise polymers, selected from polydimethyl siloxane (PDMS), cyclic olefin copolymer (COC), polymethyl methacrylate (PMMA), poly carbonate (PC) or other materials with graftable surface chemistries. In some embodiments, the buffer reconstitution substrate is made of silica, glass quartz or combinations thereof. In some embodiments, the buffer reconstitution substrate may be a quartz-based membrane or matrix. In an alternate embodiment, the glass-based matrix, such as glass fiber or glass wool may be used as buffer reconstitution substrate.
In one embodiment, the buffer reconstitution substrate is hydrophilic, which enables the membrane to wet out quickly and completely. The hydrophilic substrate eliminates the need for expensive pre-wetting treatment and increases the flow rate of the fluid passing through the substrate.
In some embodiments, the wash buffer reservoir (reagent storage location) and elution buffer reservoir are separated by a partition. In some examples, the partition may be made, for example, of a membrane, such as a metallic or polymeric strip or sheet. In some embodiments, the wash buffer reservoir and the elution buffer reservoir may each comprise one inlet and one outlet. In one or more embodiments, the wash buffer reservoir and the elution buffer reservoir may each comprise one inlet, wherein both of the inlets are connected to one conduit, which may be further connected to the downstream EOP.
In some embodiments, an area of a substrate containing impregnated wash buffer reagent is separated from the rest of the substrate by a membrane or partition, or is enclosed in a chamber. The area is referred in this example as a wash buffer reservoir. In some embodiments, an area of a substrate containing impregnated elution buffer reagent is separated from the rest of the substrate by a membrane or partition, or is enclosed in a chamber. The area is referred to in this example as an elution buffer reservoir. The wash buffer and elution buffer reservoirs may be coupled to other parts of the device through conduits. The conduits have an inlet and an outlet to the reservoirs. The outlet for wash and elution buffer reservoir may be different. Each of the reservoirs may comprise at least one outlet, wherein the outlets from both the reservoirs may be connected to one or more conduits, which are further connected to the extraction matrix through one or more valves. The two reservoirs may have two outlets, wherein the outlets are connected with one common conduit, which opens to the substrate or extraction matrix.
The wash buffer reagents may be present in the substrate in a dried, semi-dried or wet form. The wash buffer reagents are required to be hydrated by a buffer solution, water or any solvent, wherein the reagents are present in the dried form. In some embodiments, the reagents are rehydrated before use for washing the matrix. The hydration is also required, when the reagents are in semi-dried condition. After hydration, the reagents are dissolved in a buffer or solvent forming a wash buffer solution followed by transfer of the solution to the extraction matrix.
In some embodiments, the wash buffer reagents may comprise a detergent or chaotrope, that reduces various intra or inter molecular interactions between different organic or inorganic molecules, cell debris, lipids, proteins and the interactions of the one or more of them with the matrix. The wash buffer may remove the cell debris, excess lytic reagents or other impurities from the matrix after cell lysis, leaving the nucleic acids attached to the matrix. The wash buffer may further comprise one or more stabilizing agents or chelating agents, such as EDTA, which is used for nucleic acid stabilization.
The elution buffer reservoir may comprise elution buffer reagents impregnated in the matrix. In one or more embodiments, the elution buffer reagent may comprise TE buffer. In one embodiment, 1×TE buffer with 0.1% Tween is dried on cellulose paper as elution buffer reservoir. Elution and storage of the nucleic acids in TE buffer is helpful if the EDTA does not affect downstream applications. EDTA chelates divalent ions, such as magnesium, which may be present in the purified nucleic acids. The EDTA inhibits contaminating nuclease activity, as the divalent cations function as a cofactor for many of the nucleases under certain conditions. In one or more embodiments, the elution buffer reagent is present in the substrate in a dried, semi-dried or wet form. The reagents may be hydrated or rehydrated before eluting the nucleic acids from the matrix.
The device may comprise a self-rupturing component, as shown in
The self-rupturing component may comprise polymer, glass, silicon, metal or combinations thereof. The chamber-seal may be a heat-sealed thermoplastic, hot-melt adhesive, seal formed by an ultrasonic bonding, induction foil seal, or any other sealing method known in the art to maintain hermeticity until the sealing membrane opens or ruptures on a threshold pressure.
An orthogonal view of a self-rupturing foil-sealed chamber 16 is shown in
Referring to
In some embodiments, the sealed-chamber is ruptured by a high internal pressure. The pressure may be released through the chamber-seal which subsequently releases the liquid from the chamber. In some embodiments, the use of a controlled pressure source enables a liquid to flow at a steady flow rate. In one embodiment, the pressure source may be a high pressure generating EOP using low applied voltage, wherein the EOP may either be powered with a small battery source or using a battery free EOP. The pressure source, such as an EOP, is operationally coupled to the chamber-seal directly or indirectly. In some embodiments, the pressure source, such as an EOP, is operationally coupled to the chamber-seal through a small channel that controls the pressure at the seal during rupturing. The high pressure, e.g. a pressure of equal to or more than 1 PSI generated by the EOP, ruptures the chamber-seal of the sealed self-rupturing component.
The EOP may be activated by an external or internal power source 35, depicted as 51 in
Once the self-rupturing component, such as a sealed chamber is ruptured, the pressure source, such as an EOP is used to retain control over the release of the stored liquid, allowing reversible control over flow rates in and out of the chamber. An EOP-controlled, liquid-filled sealed-chamber may be operationally coupled to the sample inlet, wherein the EOP actuation results in a negative pressure exerted on the sample to control intake into the device. The EOP-controlled rupture and release of liquid from the hermetically sealed chambers may allow temporal control of the buffer exchange or reconstitution. The control of buffer release optimizes concentration of the buffer before reaching to the substrate, such as an extraction matrix. The liquid flow and the reconstitution rate may be controlled by varying the voltage or current applied across the EOP element.
Unlike mechanically or externally ruptured foil-sealed chambers, an internally ruptured or EOP-actuated sealed-chamber allows liquid flow in both directions 53, as shown in
In some embodiments, an EOP-controlled, liquid-filled sealed chamber is operationally coupled to a second chamber comprising a liquid. In this embodiment, the foil-sealed chamber is replaced with a flexible membrane component, 39, as shown in
The fluid is applied to the substrate for washing or elution, wherein the fluid is drawn from one or more reservoirs, which are externally located to the substrate-integrated device. In some embodiments, the MFM device is operatively connected to at least one external reservoir comprising one or more fluids. In one embodiment, the pumping liquid or fluid or working solution for EOP is stored in the external reservoir. In one embodiment, the fluid stored in the external reservoir may be a buffer, water or other solvent. In some embodiments, the fluid has a pH from about 3.5 to 8.5. In an alternative embodiment, the pumping solution is a borate buffer with a pH of about 7.4 to 9.2 and an ionic strength between about 25 to about 250 mM.
In one or more embodiments, the biological waste materials or other impurities are collected after washing the substrate. The collection chamber for collecting biological waste and other impurities may be a part of the device or may be externally located from the device. The container may be a chamber, vessel, bag or disposable. The container for collecting waste may be adopted for easy removal and integration with down-stream analytical processes. The biological waste may contain tissue fragments, cell debris, lipids, excess reagents or other impurities.
Similarly, a container for collecting eluted nucleic acids may also be incorporated. The container may be a chamber, vessel, bag or disposable. In addition, the container for collecting purified nucleic acid, may be adopted for easy removal and integration with down-stream analytical processes. In one or more embodiments, the container is coupled to the device for collecting the purified nucleic acids. The container may be coupled to the device directly or indirectly, using one or more conduits or adapters.
The isolation of nucleic acids from biological material is carried out using the MFM device, the biological materials used in the embodiments may comprise a physiological body fluid, a pathological body fluid, a cell extract, a tissue sample, a cell suspension, a liquid comprising nucleic acids, a forensic sample and combinations thereof. In some embodiments, the biological material is a physiological body fluid or a pathological body fluid, such as the fluid generated from secretions, excretions, exudates, and transudates, or cell suspensions such as, blood, lymph, synovial fluid, semen, saliva containing buccal swab or sputum, skin scrapings or hair root cells, cell extracts or cell suspensions of humans or animals. In some embodiments, the physiological/pathological liquids or cell suspensions may be extracted from plants. In one or more embodiments, the extracts or suspensions of parasites, bacteria, fungi, plasmids, or viruses, human or animal body tissues such as bone, liver or kidney. The biological material may also include a liquid comprising DNA, RNA and combinations thereof, mixtures of chemically or biochemically synthesized DNA or RNA. The device may be portable or field-able, so that the biological materials can be collected from any place and load to the device to isolate nucleic acids under reduced degradation condition for faster downstream analysis.
In some examples, the nucleic acids are extracted using an MFM device that runs on small batteries for use as hand held devices. The MFM device may comprise a self-contained (battery-free) EOP without any external power source. In one embodiment, the MFM device is packaged with a power source, wherein the entire assembly may be self-contained. In such embodiments, the MFM device is a portable, field-able, simplified, user friendly device to operate and carry as per the user need.
In some embodiments, the device provides a storage facility for nucleic acids. The nucleic acids may be stored for at least eight to ten hours, if the downstream application facility is not instantly available. For example, the nucleic acid is required to store for few hours, when the nucleic acid is isolated from a blood sample collected from a field and the downstream application facility is situated in a distant location.
In some embodiments, the methods and devices for extracting nucleic acids may be adapted for lab-on-a-chip devices and for applications, such as drug delivery, liquid drug delivery, biochemical analysis, genomics, proteomics, healthcare related applications, defense and public safety applications; medical applications, pharmaceutical or biotech research applications, environmental monitoring, in vitro diagnostic and point-of-care applications, or medical devices. They can be adapted for other applications including, but are not limited to, DNA amplification, DNA purification, PCR or real time PCR on a chip, or adaptive microfluidic mirror arrays.
In one or more examples, the methods and devices may be fully or partially automated. Automation may be required to reduce human intervention during extraction and purification of the nucleic acids. The use of an automated device further helps to minimize contamination during nucleic acid purification from various biological samples. A fully automatic device is desirable for forensic applications, wherein the objective is to purify nucleic acids from a trace amount of sample. An externally located controller may be operationally coupled to the device to drive the system, excluding any manual intervention after application of the biological sample to the device or sample inlet.
In some embodiments, the method may be employed using a device, which is further configured to integrate with a system, more specifically with an analytical system. As noted, the device may have one or more attachments through which the device may integrate with another system depending on the requirement. One or more adapters may be used to couple the device with another system. In one embodiment, the adapter has a holder to hold the device and a connecter for connecting to the system. In some embodiments, an adapter may be attached to the device, wherein the adapter has at least two holders for holding the device and the system on it, and thereby couple the device with the system. For example, an adapter is used for coupling the MFM device with a downstream analytical system. In some embodiments, the device itself is configured to have one or more holders, connecting ports or combination thereof, which mechanically couples the device to another system. The device may be electronically coupled to another system for downstream applications.
As noted, the device is configured to integrate with a system, the system may be a microfluidic system or a conventional analytical system. A schematic representation of a system is depicted in
One embodiment of the system 62 is shown in
Materials: Solid phase extraction matrices used for the experiments, include 31-etf cellulose (GE-Whatman, UK), QMA quartz fiber membranes (GE-Whatman, UK), and GF-A or GF-C glass fiber membranes (GE-Whatman, UK). Illustra™ spin column (from GE Healthcare) was used for testing various matrices, reagents, buffers, and standardizing nucleic acid purification protocol. Illustra™ microspin column also served the purpose of control experiments or used as a control device as compared to the MFM chip (device of the invention) for different experiments. Illustra PuRe Taq Ready-to-Go™ PCR beads (from GE Healthcare) was used for DNA amplification using PCR.
A number of matrices were used to compare the properties of matrices with respect to sample loading capacity. A larger sample size may be accommodated by eliminating the drying step and using the EOP to drive the sample through multiple quartz-based matrices. The yield of DNA using two different sample volumes applied to the quartz-based matrix was determined Experiments were performed for 20 μL, 70 μl, and 500 μl sample volumes, and the yield of DNA was shown to decrease with increasing sample sizes. The DNA yield and concentration was measured using a fluorescent Picogreen Assay. Yields approaching 50% were obtained from single matrices at the lower input volumes (70 μL; see
Similarly, loading capacity of different matrices was also determined by comparative analysis of load capacity using cellulose, glass and quartz matrices. For this experiment, quartz matrix QMA cellulose matrix 31 ETF and glass matrix GF-A in spin column were used. An aqueous sample was added to each of the matrices, wherein the DNA was purified from the sample load and the loaded volume was compared, wherein the quartz matrix shows maximum capacity for sample load (data not shown).
A number of matrices with different chaotrope/detergent solutions were used to compare the properties of matrices with respect to nucleic acid retention, isolation of nucleic acids from a complex sample, or loading capacity.
DNA yield using glass and quartz matrices were compared using multiple chaotrope/detergent combinations. Whatman™ Glass microfiber grade A (GF/A) was used as glass matrix, which is known for fine particle retention, high flow rate, as well as good loading capacity. The glass fibers were used in Illustra™ columns.
150 ng of E. coli cell extract was loaded on to each of the matrices. The lytic components of the matrix were rehydrated by the sample and non-nucleic acid materials were removed during the first washing step. Nucleic acids, such as DNA was eluted off from different types of matrices (sub-components of the MFM) using two different chaotropes KI and GuSCN in final concentration of 5 M. The combination of the weaker chaotrope (KI) and quartz-based membranes consistently showed the highest elution rates. The glass matrix in Illustra® column provided yields of 48% DNA, while the combination of quartz matrix and the KI chaotrope provided yield near 70% yield of DNA, as shown in
A sample of E. coli from an overnight culture was loaded on to the solid phase extraction (SPE) matrix of the MFM device, and dried for 30 minutes to ensure cell lysis. The SPE was impregnated with the cell lysis reagents, resulting in extraction of the nucleic acids, which bound to the SPE matrix. In the washing cycle, a 70% ethanol wash was passed through the SPE matrix to wash away the cell debris and other materials except bound nucleic acids, using a normal syringe pump. The washing step was repeated for five times. The wash liquid (a liquid after washing the impurities from the matrix) for each wash was collected in different tubes. The wash liquids were collected after five washes and were analyzed to determine presence of DNA using a Picogreen® fluorescence assay.
In another experiment, an E. coli specific PCR was performed using DNA from an E. coli spiked mouse blood. In this case, the mouse blood was mixed with E. coli cell extracts and loaded on to the SPE matrix. The quartz/KI combination was used for isolating E. coli DNA from the complex sample using the method described above. The eluted DNA was subjected to E. coli specific PCR analysis. PCR was performed using Illustra PuRe Taq Ready-to-Go™ PCR beads using E. coli specific primers (SEQ. ID. No 1: 5′ TTAAAGTCTCGACGGCAGAAGCCA 3′ and SEQ. ID. No 2: 5′ AACATCTTTCATCAGCTTCGCGGC 3′). In the PCR, one amplicon was employed having SEQ. ID. No 3: 5′-TTAAAGTCTCGACGGCAGAAGCCAGGGCTATTTTACCGGCGCAGTATCGC CGCCAGGATTGCATTGCGCACGGGCGACATCTGGCTGGCTTCATTCACGC CTGCTATTCCCGTCAGCCTGAGCTTGCCGCGAAGCTGATGAAAGATGTT-3′. In
The purification of substantially intact form of the nucleic acids is also enabled as shown in
Nucleic acids that were eluted using the traditional heating methods for cellulose storage membranes at higher temperature (95° C.) showed degraded nucleic acids, as shown in lane 7 of
While only certain features of the invention have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the scope of the invention.
This application is a continuation-in-part of U.S. patent application Ser. No. 13/562,947 entitled “Devices and Systems for Isolating Biomolecules and Associated Methods Thereof”, filed Jul. 31, 2012; which is herein incorporated by reference.
This invention was made with Government support under contract number HDTRA1-10-C-0033 awarded by the Defense Threat Reduction Agency. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13562947 | Jul 2012 | US |
| Child | 13599124 | US |
