Patent Application
20110272282
The invention relates to the induced movement of particles such as proteins and other molecules through media such as gels and other matrices. Some embodiments provide methods and apparatus for selectively concentrating particles of interest. Some embodiments relate to scodaphoresis methods and apparatus.
Scodaphoresis (or “SCODA”) is an approach that may be applied for concentrating and/or separating particles. SCODA may be applied, for example, to DNA, RNA and other molecules. The following background discussion of SCODA is intended to provide examples that illustrate principles of SCODA and is not intended to impose any limitations on the constitution, makeup or applicability of SCODA methods and apparatus generally.
SCODA is described in:
SCODA can involve providing a time-varying driving field that applies forces to particles in some medium and a time-varying mobility-altering field that affects the mobility of the particles in the medium. The mobility-altering field is correlated with the driving field so as to provide a time-averaged net motion of the particles. SCODA may be applied to cause selected particles to move toward a focus area.
Some modes of SCODA exploit the fact that certain particles in appropriate media exhibit non-linear responses to electric fields. In such modes, suitably time-varying electric fields can cause certain types of particles to be focused or concentrated at locations within the medium. In many practical cases, the instantaneous velocity of a particle in a medium under the influence of an electric field is given by:
{right arrow over (v)}=μ{right arrow over (E)} (1)
where {right arrow over (v)} is the velocity of the particle, {right arrow over (E)} is the electric field and μ is the mobility of the particle in the medium given, at least approximately, by:
μ=μ0+κ|{right arrow over (E)}| (2)
where μ0 and κ are constants. Particles with larger values for κ tend to be focused more strongly than particles with smaller values for κ.
In some cases, SCODA is performed by providing an electrical field having a rotating component and a quadrupole perturbation. The rotating component may be specified, for example, by:
E
x
=E cos(ωτ) (3)
and
E
y
=E sin(ωτ) (4)
where E is a magnitude of the electric field component that rotates at an angular frequency ω, and Ex and Ey are respectively x- and y-components of the rotating electrical field. The perturbing quadrupole component may be specified, for example, by:
dE
χ
=−dE
x
x cos(2ωτ) (5)
and
dE
γ
=dE
q
y cos(2ωτ) (6)
where dEx and dEy are respectively x- and y-components of the perturbing electrical field, x and y are distances from an origin at the center of the quadrupole field pattern and dEq is the intensity coefficient of the perturbing quadrupole field. With this SCODA field, the average radial velocity of a particle toward a focus location can be shown to be given by:
where {right arrow over (r)} is a vector pointing toward the focus location and having a magnitude equal to the distance of the particle from the focus location.
The size of a spot into which particles can be focused depends upon κ as well as on the rate at which the particles can diffuse in the medium as follows:
where R is a radius of the focused spot and D is a diffusion coefficient.
Molecules having large values of √{square root over (κ/D)} may focus in the medium under SCODA conditions, and are selectively concentrated within smaller radius distances R relative to molecules with smaller values of √{square root over (κ/D)}.
A limitation of SCODA applications in which electric fields are applied to inject target molecules from a sample into a gel or other SCODA medium is that the applied electric fields can cause electrical currents. The more electrically conductive the sample, the larger are the electric currents generated for a given electrical field strength within the sample. The electric currents result in heating which can damage target molecules and/or the SCODA medium. Excessive heating can also impair the efficacy of SCODA.
This invention has a number of aspects that may be applied independently or in combination with one another.
In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed description.
Example embodiments are illustrated in the accompanying drawings. The illustrated embodiments are intended to be illustrative and not restrictive.
In the drawings, identical reference numbers identify similar elements or acts. The sizes and relative positions of elements in the drawings are not necessarily to scale. For example, the shapes of various elements and angles are not drawn to scale, and some of these elements are arbitrarily enlarged and positioned to improve drawing legibility.
In the following description:
Unless the context clearly requires otherwise, throughout the specification and claims which follow:
As noted above, where the mobility μ of a type of particle is given, at least approximately, by Equation (2) particles of types having larger values for κ tend to be focused more strongly than are particles of types having smaller values for κ. κ may be described as a ‘non-linearity coefficient’ or a ‘coefficient of field dependence of the particle's mobility’. One aspect of the invention provides SCODA methods and devices in which the value of κ for target particles is increased. In some embodiments, the target particles are biomolecules. In some specific embodiments, the target particles comprise one or more proteins. In some embodiments, the SCODA driving and mobility-altering fields comprise electrical fields.
This aspect provides one or more process steps that alter κ for target particles. The process steps comprise one or more of:
Such process steps can alter physical properties of particles (which may be molecules, for example). The altered properties that contribute to the alteration of κ may include one or more of (but are not limited to): electric charge, shape, degree of folding, drag, and conformation.
One example of a physical process step that can increase κ for a target particle is heat treatment. The heat treatment may include, for example, heating a sample to a temperature and for a period of time sufficient to cause a change in target particles in the sample. In some embodiments the sample is brought to a boil or is heated by thermal contact with a boiling water bath. Heating can be particularly effective for altering κ where the target particle is a protein or other molecule that becomes denatured and/or experiences a change in the degree of folding as a result of the heating.
Examples of a chemical process step that can increase κ for a target particle are treatment with chemicals that are effective to impart a net electric charge to target particles and/or alter a configuration of the target particles. In some embodiments the target particles are molecules and the chemical treatment denatures and/or changes the degree of folding of the target particle molecules.
The chemical treatment may include, for example, treatment with one or more of: tris-glycine, dithiothreitol, and sodium sodecyl sulfate. In some embodiments the target particles comprise disulfide bonds and the chemical treatment comprises treatment with a chemical that breaks disulfide bonds. In some embodiments the chemical treatment comprises treatment with a detergent such as a suitable anionic surfactant.
Molecules or other particles may be affixed to target particles in various ways. For example, “handle” molecules, having a specific response to SCODA fields, may be attached to “target” molecules by one or more of:
Target particles to which handle molecules may be attached may comprise, but are not limited to, biomolecules such as proteins, enzymes and nucleic acids such as RNA and DNA. In some example embodiments the handle molecules comprise nucleic acids or proteins (the proteins may be modified so as to be readily focused by a SCODA field). In some embodiments the handle molecules comprise a marker such as a dye or the like.
In some embodiments the handle particles or a linking agent provided to link handle particles to target particles have a specific affinity for particular target particles. For example:
Where the handle particles have an affinity for target particles, the handle particles may be attached to the target particles by mixing handle particles into a sample containing the target particles. For example, where the target particles comprise a particular protein, the handle particles may comprise a strand of nucleic acid (e.g. DNA or RNA) linked to an antibody that binds to the protein. The antibody-linked nucleic acid can be mixed with a sample containing the protein targeted by the antibody. The resulting sample can then be processed with SCODA to concentrate the targeted protein at a point in a medium. Such Focusing may occur even in cases where the protein itself is electrically neutral or, for some other reason, is not focused very much or at all by the applied SCODA fields.
The foregoing techniques may be applied to improve the selectivity of SCODA Focusing for selected target particles and/or to improve the degree to which SCODA focuses target particles. In some embodiments, two or more of the above techniques are applied. For example, in one embodiment a sample is prepared for SCODA by a physical or chemical treatment step which alters target particles followed by a process step which selectively attaches handle particles to the altered target particles. The altered target particles are then concentrated by SCODA.
Under suitable preparation/lysis conditions, SCODA may be applied to concentrate target particles such as biomolecules (e.g. molecules of nucleic acid, proteins, enzymes and the like) from a wide range of samples. The samples may include, for example, human or animal samples including: blood, tissue, urine, stool, hair, biopsy, sputum, lavage fluids, discharge, mucus, skin; environmental samples such as: food, water, soil, collected aerosols, plant samples; archeological samples such as: bone, fossil, tar sands, tar pit, ice cores; and so on.
In some embodiments, target particles are given a selected value for the parameter κ/D by one or some combination of physical treatment steps, chemical treatment steps and affixation of handle particles and then the target particles are separated from other particles on the basis of differences in the parameter κ/D.
The above techniques are useful for enhancing SCODA focusing of proteins. Protein molecules, including fragments of proteins, tend to have relatively low net electric charge and are typically folded in a way that limits the amount of conformational change that results from changes in the strength of applied electric fields. Consequently, proteins tend to have low values for x and tend not to focus very well under electrophoretic SCODA.
Separation and/or concentration of a protein in electrophoretic SCODA may be facilitated by subjecting the protein to one or more physical and/or chemical treatments that increase the value of κ. In some embodiments these treatments denature the proteins and cause the net electric charge on molecules of the protein to increase. Samples containing proteins may comprise, for example, total protein from a cell, or group of cells such as a cell culture. In some embodiments samples containing proteins are prepared according to a protocol for preparing samples for sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).
The SCODA separation and/or concentration of the protein may also be improved by coupling the protein to another molecule (a ‘handle’), such as DNA or RNA, that has a significantly greater value of κ than the protein. In some cases the aggregate particle (protein attached to handle molecule) also has a smaller value for D than the protein alone. By increasing κ/D these techniques facilitate separation and concentration of proteins by electrophoretic SCODA. Handle molecules may be attached to native proteins or protein fragments or to proteins that have been pretreated by one or more physical and/or chemical treatments as described above, for example.
A sample was prepared using pre-stained protein molecular weight marker commercially available from New England Biolabs Inc. (‘NEB’) of Ipswich, Mass., United States of America. The marker included proteins in several different molecular weight bands ranging from 6.5 to 175 kDa. The proteins in the marker were covalently coupled to a bromophenol blue dye which makes the protein bands visible in an electrophoretic gel under white light.
100 μL of NEB pre-stained Protein Marker, Broad Range (NEB #P7707s) was treated with 90 μL 5×Tris Glycine solution (125 mM Tris-Cl, 1.25M Glycine, 0.5% SDS), 10 μL 800 mM Dithiothreitol (‘DTT’) and 10 μL 10% SDS. The sample was heated by submersion in a boiling water bath for 3 minutes to denature the protein, and then added to 600 μL of distilled H20. It is thought that: the heating denatures proteins in the sample, SDS binds to the denatured proteins imparting negative charges to the protein molecules, and DTT prevents rebinding of disulphide bonds in the protein reducing the secondary structure of the protein. The sample was then loaded into sample chambers 120, 130 of SCODA apparatus 100.
In this experiment, gel 110, was a 3.5% polyacrylamide (29:1 crosslink ratio) gel prepared with 0.15×TBE buffer. Gel 110 was 16 mm wide and was prepared using a custom gel cap and side dams to limit oxygen exposure of the acrylamide and enhance polymerization.
In this experiment the power supply was configured to apply voltages as set out in
In this experiment, the voltages at Time 1 were applied for 0.5 s, Time 2 for 1 s, Time 3 for 0.5 s, and Time 4 for 1 s, for a total run time of 1.5 hrs. The applied voltages drive protein molecules into gel 110 and concentrate protein molecules in gel 110. The voltages of
It was found that, under the influence of the SCODA fields, the pre-stained protein molecular weight markers from the samples loaded in sample chambers 120, 130, were focused in a region of gel 110. Gel 110 is free from protein in
A portion of gel 110 including location 160 was cut out. A DC electrophoresis analysis of the extracted sample was performed using an SDS-PAGE gel. Control samples consisting of the ladders which comprised the original sample were also run on the SDS-PAGE gel.
This result demonstrates that electrophoretic SCODA may be applied to concentrate proteins and also that electrophoretic SCODA may be applied to separate longer proteins from shorter proteins.
Injection of Protein-Containing Samples into SCODA Media
In some embodiments, target particles are injected into a SCODA medium, such as a suitable gel, by techniques including electrokinetic injection, quadrupole injection and the like which use applied electrical fields to cause target particles to move from a sample into the SCODA medium. Such techniques can be inefficient or may not work at all in cases where the target particles are electrically neutral or have only small net electrical charges.
In one embodiment, a sample containing proteins which it is desired to concentrate is pre-treated as described above. The pre-treatment causes the target proteins to respond to the electrical fields applied to inject the target proteins into a SCODA medium. For example, injection and concentration of positively charged proteins from a sample may be achieved using combined electrokinetic injection, as discussed in PCT Publication No. 2006/081691 and suitable SCODA fields.
When apparatus like that in
With the
Focusing Two or More Types of Target Particle from a Sample
In some embodiments, two or more different groups of target particles from a single sample may be focused at different locations. In some embodiments the two groups of target particles are focused simultaneously. In other embodiments different groups of target particles are focused sequentially.
The different groups of target particles may have different charges. For example, particles of the first group of target particles may be positively charged while particles of a second group of target particles may be negatively charged.
The first group of target particles may comprise, in some embodiments, a protein which has been pre-treated to have a positive electrical charge. For example a sample may be pre-treated with cetyl trimethylammonium bromide (CTAB) such that a net positive charge is given to protein molecules in the sample. The second type of target particles may comprise, in some embodiments, a protein which has, or has been pre-treated to have, a negative electrical charge. For example, a sample containing proteins may be treated with SDS such that a net negative charge is given to protein molecules in the sample. Optionally, “handle” molecules are attached to one or both of the groups of target particles. The handle molecules may carry a net charge. For example, the handle molecules may comprise DNA or RNA. The DNA or RNA may have a net negative charge. In some embodiments, one or both of the first and second groups of target particles comprise nucleic acids such as DNA or RNA.
Additional electrodes 475, 485, 495 are in contact with buffer reservoirs 470, 480, and 490. SCODA electric fields may be provided in second gel 420 by applying suitable voltages to electrodes 435, 475, 485, and 495. This may be achieved by connecting electrodes 435, 475, 485 to suitable channels of a programmable power supply, for example. Under the influence of SCODA fields, positively charged target particles in the sample contained in chamber 430 may be injected into gel 410 and may be focused in a region of gel 410, and negatively charged particles from the sample may be simultaneously injected into gel 420 and focused in a region of gel 420.
In some embodiments, a DC bias voltage is applied across injection chamber 430 to drive positively charged particles in one direction while simultaneously driving negatively charged particles in the opposite direction. Such a DC bias voltage may be applied, for example, by making electrode 455 negative relative to electrode 485.
In a further embodiment, quadrupole injection may drive the first group of target particles from injection chamber 430 into gel 410 and quadrupole injection may drive the second group of target particles into gel 420.
As an example application, consider the case where a sample in sample chamber 430 contains both DNA and a protein. Such a sample may be obtained, for example by lysing a bacteria culture. In an illustrative example, a sample containing DNA and proteins could be created by lysing 0.5 mL of E. coli DH10B culture with CTAB-Bacterial Lysis Buffer (CTAB-BCB). CTAB-BCB may comprise 10 mM Tris HCl, 100 mM Na EDTA, 20 mM mercaptoethanol and 2% CTAB (Cetyl trimethylammonium bromide). In this illustrative example, treatment with CTAB may yield a sample solution containing positively-charged proteins and electrically-neutralized DNA. An amount, for example, 100 μL of the solution may be added to sample chamber 430 of apparatus 400. The electrically charged protein may then be injected into gel 410 by way of electrokinetic injection while the electrically neutral DNA remains in sample chamber 430. The protein may be concentrated at a location in gel 410 by scodaphoresis.
Subsequently, the DNA may be treated so that the DNA acquires an electrical charge. This treatment may be performed in sample chamber 430. For example, 50 μL of 20% SDS may be added to sample chamber 430. The DNA may become negatively charged as a result of interaction with the SDS. Electrokinetic injection may then be used to cause the DNA sample to enter gel 420. The DNA may be concentrated at a location in gel 420 by scodaphoresis.
Negative and positively charged particles may be injected with either quadrupole injection fields and electrokinetic injection fields. The above embodiments are merely exemplary. Other quadrupole injection/electrokinetic injection and SCODA fields may be used to inject and concentrate sample outputs within apparatus 400. Further, apparatus 400 may have additional or fewer sample chambers and buffer reservoirs. In some embodiments, additional electrodes may be present in apparatus 400. In some embodiments, three or more gels or other SCODA media are arranged around a common sample chamber.
There are cases in which it is desirable to use SCODA to concentrate target particles from samples that have relatively high electrical conductivity. For example:
As a non-limiting example, the conductivity of a SCODA medium may be chosen to have an electrical conductivity that is high enough that electric fields in one or more sample chambers adjacent to the SCODA medium are sufficient to inject target particles into the SCODA medium in a desirably short time. If the electrical conductivity of the SCODA medium is very low then most electrical potential may be dropped across the SCODA medium resulting in undesirably small electric injection fields and undesirably long injection times. On the other hand, the electrical conductivity of the SCODA medium is desirably sufficiently low that applied SCODA electric fields do not cause too much heating of the SCODA medium. In some embodiments the SCODA medium comprises a gel having an electrical conductivity of 250 μS/cm. In some embodiments, the SCODA medium has an electrical conductivity of a few hundred μS/cm or lower (e.g. 200 to 800 μS/cm or less). In some embodiments the SCODA medium has an electrical conductivity of 300 μS/cm or less. For fast efficient injection of target particles into the SCODA medium it is desirable that the sample have an electrical conductivity that is smaller than that of the SCODA medium.
Embodiments provide SCODA methods which include a step for reducing electrical conductivity of a sample containing target particles. In some embodiments, the conductivity-reducing step comprises a buffer exchange step performed prior to injection of target particles into a SCODA medium. The buffer exchange step transfers the target particles from a sample having higher electrical conductivity to a sample having a lower electrical conductivity such as a low salinity buffer. In some embodiments the buffer exchange step is also effective to lyse cells in the sample and preferentially extract nucleic acids or other biomolecules of interest. In some embodiments the conductivity-reducing step comprises a step which removes or neutralizes charge carriers (such as salts) from a sample which also contains target particles. Removal or neutralization of the charge carriers reduces electrical conductivity of the sample. In some embodiments the conductivity-reducing step reduces electrical conductivity of the sample to a level that is on the order of or less than the electrical conductivity of the SCODA medium.
In some embodiments a buffer exchange step comprises placing a sample containing target particles in contact with a solid material to which target particles bind, separating the solid material from the remaining sample fluid, placing the solid material and associated target particles in contact with a low-conductivity buffer, and transferring the target particles into the low-conductivity buffer.
For example, where the target particles comprise DNA, the buffer exchange step may comprise incubation of a high salinity sample containing DNA in the presence of a DNA-binding matrix such as diatomaceous earth or silica gel under chemical conditions that cause nucleic acids to bind to the DNA-binding matrix. Once binding has occurred, the high salinity sample is separated from the DNA-binding matrix and a low salinity buffer is added. The DNA is caused to unbind from the DNA-binding matrix by providing suitable conditions in the low-salinity buffer. For example, under high pH conditions DNA unbinds from diatomaceous earth. The low salinity buffer, with or without the DNA-binding matrix, is placed in a SCODA sample chamber. SCODA DNA extraction and concentration is then performed.
In some embodiments, DNA is recovered from the DNA-binding matrix by washing the matrix to remove contaminants, as well as any remaining high-conductivity material from the sample. After washing, elution of the DNA with a suitable low ionic strength elution buffer or water, under suitable conditions (e.g. neutral or slightly basic pH), may be completed. The elution buffer may be ideally suited for SCODA and therefore the solid-phase step acts as an ideal buffer-exchange step in the SCODA system, converting the sample from high salinity buffer to low-salinity buffer.
The combination of SCODA with solid-phase DNA extraction can provide advantages over other DNA separation and concentration methods. Because SCODA can extract target particles from relatively very dilute samples, a relatively large amount of matrix can be used to bind DNA or other target particles. This is not practical where DNA separation is performed by other methods which cannot handle large elution volumes. Additionally, SCODA may reject certain contaminants that carry through the solid-phase purification.
Standard purification methods using DNA-binding matrices, such as silica gel, are limited to using small amounts of matrix because the matrix volume determines the final elution volume, which must be small to avoid excessively low concentration of the eluted product. In addition, elution occurs through fluid flow which limits elution time and elution efficiency. Also, contaminants often carry through such methods due to indiscriminate binding to the matrix.
At block 601, a highly conductive sample, such as a lysate containing 4M guanididium, is added to an amount of DNA-binding matrix (such as diatomaceous earth or silica gel). In some embodiments, a large amount of DNA-binding matrix may be used, such as several millilitres or several hundred microlitres. This is not mandatory, however. In other embodiments, smaller amounts of DNA-binding matrix may be used.
At block 602, DNA present in the highly-conductive sample is allowed to bind to the DNA-binding matrix.
At block 603, the DNA-binding matrix is separated from the highly-conductive sample. In some embodiments block 603 comprises centrifugally separating the matrix from the sample. For example, the sample and matrix may be introduced into a spin column from which fluid may be spun out by a centrifugal system. In further embodiments, fluid may be separated from the matrix by filtering, by means of a gravity flow system, positive pressure system, or a vacuum manifold.
Block 604 provides an optional washing step. For example, block 604 may comprise washing the separated matrix with an ethanol containing buffer. Such a buffer may wash salt and other contaminants from the matrix.
At 605, a low salt elution buffer is brought into contact with the matrix to elute the DNA from the matrix. In some embodiments, block 605 comprises using a relatively large amount of low salt elution buffer (for example several millilitres or several hundred microlitres). In an example embodiment, the elution buffer has a volume on the order of 5 mL. In some embodiments, the volume of the elution buffer is substantially the same as a capacity of a SCODA sample chamber. For example, the SCODA sample chamber may have a capacity of a few mL (e.g. 5 mL) and in block 605 the target particles may be eluted into a few mL (e.g. 5 mL) of a low-salt buffer. In other embodiments smaller amounts of low salt elution buffer may be used.
At 606, the eluate is purified and concentrated by SCODA. The SCODA can proceed more quickly and efficiently and with less heating than would be possible if one attempted to perform SCODA by placing the highly conductive unprocessed starting sample in a sample chamber of SCODA apparatus like that described above (for example sample chamber 120 and/or 130 shown in
To prepare gel 700, 100 μL of 5 ng/μL pUC19 DNA in TE buffer was mixed with 400 μL of distilled H20, 575 μL of 8M Guanidine HCl and 75 μL of 200 mg/mL diatomaceous earth in a microcentrifuge tube. This mixture was vortexed vigorously for 30 s and spun down on a microcentrifuge for 30 s. The supernatant was removed, being careful not to disturb the binding matrix, and 1000 μL of 95% ethanol was added to the tube. The mixture was then vortexed for 30 s and spun down for 30 s, and the supernatant carefully removed. The final elution involved addition of 1 mL TE buffer, vortexing for 30 s, spinning down for 30 s and removal of the supernatant, repeated twice. The resulting buffer was then diluted to 5 mL with distilled H20 and loaded directly into a sample chamber of SCODA apparatus. In the run resulting in column 703 the diatomaceous earth was introduced into the sample chamber. In the run resulting in column 702 the diatomaceous earth was removed before the diluted supernatant was introduced into the SCODA sample chamber.
The SCODA medium in each case was a 1% low melting point agarose SCODA gel in 0.25×TBE. The DNA was injected into the SCODA medium using an electric field of 20V/cm for 20 minutes. The DNA was then concentrated to a tightly focused spot using 50V/cm SCODA fields having a 4s rotational period for 2 hrs. The focused DNA was extracted from the SCODA gel and run in a DC gel against a control 701 to confirm DNA recovery, shown in
To extract plasmid DNA from E. coli, 5 mL of overnight culture was spun down to form a pellet of cells, and supernatant removed. 400 μL of 1×TE was added to the resulting pellet, and 575 μL of 8M Guanidine HCl and 75 μL of 200 mg/mL diatomaceous earth were added as well. The mixture was then vortexed vigorously for 30 s after which it was allowed to sit at room temperature for 1 hr to allow lysis to proceed. After 1 hr the mixture was vortexed again for 30 s, spun down and supernatant removed. The DNA was then eluted in 1 mL of 1×TE, vortexed for 30 s and spun down. 1 mL of the solution containing the eluted DNA was loaded into a sample chamber adjacent to a SCODA gel. The sample was eluted again with 1 mL 1×TE, vortexed, and the resulting mixture, including diatomaceous earth, loaded directly into SCODA with the previous 1 mL sample. The SCODA gel comprised a 1% low melting point agarose SCODA gel in 0.25×TBE.
The DNA was then injected into the SCODA gel with a 20V/cm electric field applied for 20 minutes. The DNA was concentrated using 60V/cm SCODA fields with 4 s rotational periods for 1.5 hrs to yield a tightly focused spot. The focused DNA was extracted from the SCODA gel and run in a DC gel against a control to confirm DNA recovery as shown in
At block 801, a highly conductive solution containing target particles (for example a lysate containing 4M guanididium) is passed into a desalting column. The desalting column may contain a low salinity solution into which the highly conductive sample is added.
At block 802, the sample is filtered through the desalting column. Longer molecules, such as DNA, RNA, and other target particles of interest may pass through the desalting column more quickly than salt particles. By collecting a fraction of the fluid emerging from the column which is known to contain the target particles and not collecting the fraction of the fluid which follows, the target particles are separated into a low-conductivity fluid. Block 802 may, for example, comprise taking a predetermined volume of fluid that has flows from the desalting column. As the target particles will be concentrated and further purified by SCODA, the fraction taken from the desalting column may contain contaminants which would make the fraction unusable for other concentration protocols.
At optional block 803, the runoff from the desalting column is diluted further with a volume of low salinity solution. In some embodiments, a relatively large amount of low salinity solution may be used, such as several millilitres or several hundred microlitres. In other embodiments a smaller amount, or no, low salinity solution is added.
In some embodiments, the desalting column may discharge the fraction containing target particles directly into a volume of low salinity solution or into a sample of a SCODA apparatus (e.g. sample chamber 120 or 130 of
At block 804, the low salinity solution is purified and concentrated using SCODA. As in method 600, SCODA injection and concentration can proceed more quickly and efficiently than would be the case if one attempted to inject the target particles into a SCODA medium directly from the initial highly-conductive unprocessed sample. Target particles may be injected into a SCODA medium, for example, by a quadrupole injection field or a electrokinetic injection field.
Methods 600 and 800 may be automated by providing suitable controllers which operate valves and fluid transfer devices to process samples as described above and to deliver the processed samples into a chamber from which target particles can be injected into a SCODA medium.
Embodiments which apply methods like method 600 and/or method 800 can have significant advantages over prior methods for separating DNA or other target particles from samples. These advantages may include:
Some embodiments combine features of two or more of the embodiments described above. For example, in some embodiments, the target particles are protein molecules and the embodiments concentrate the protein molecules by methods that comprise:
In other example embodiments the target particles are protein molecules and the embodiments concentrate the protein molecules by methods that comprise:
In embodiments like any of those described herein, one or more reagents such as heparin may be added to the sample prior to performing SCODA to concentrate target particles from the sample. Such reagents may help to improve DNA yield from some samples. For example, adding heparin to a sample may help to saturate binding sites on molecules that could otherwise bind to DNA and prevent DNA from injecting into the SCODA gel. Since SCODA may be able to preferentially concentrate DNA and reject heparin from the concentrated product, the addition of heparin to the sample may improve DNA injection without carrying through to the final product.
While some of the embodiments described above have particular advantages in cases where the target particles are protein molecules or fragments of protein molecules or nucleic acids such as DNA or RNA, the methods and apparatus described herein may be applied to target particles of other types that can be concentrated by SCODA, for example, nanoparticles, polystyrene particles, polysaccharides, lipids, vitamins, hormones, carbohydrates and the like.
The foregoing detailed description has set forth various embodiments of the devices and/or processes via the use of block diagrams, schematic illustrations, flowcharts and examples. Insofar as such block diagrams, schematic illustrations, flowcharts and examples contain one or more functions and/or operations, it will be understood by those skilled in the art that each function and/or operation within such block diagrams, schematic illustrations, flowcharts or examples can be implemented, individually and/or collectively. Methods, or processes set out herein, may include acts performed in a different order, may include additional acts and/or omit some acts.
Features of the various embodiments described above can be combined to provide further embodiments. To the extent that they are not inconsistent with the specific teachings and definitions herein, all of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification, including but not limited to U.S. Patent Publication No. 2009/0139867, PCT Publication No. 2006/081691 and PCT Publication No. WO 2009/094772 are incorporated herein by reference, in their entirety. Aspects of the example embodiments disclosed herein may be modified to employ features of systems, circuits and concepts disclosed in the incorporated patents, applications and publications to provide yet further example embodiments.
Any of the techniques described may optionally be applied to concentrate target particles in wells within SCODA media as described, for example, in PCT Publication No. WO 2009/094772. In such embodiments, target particles may enter a fluid in a well and may be withdrawn by extracting fluid from the well.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope.
For the purpose of the United States of America, this application claims the benefit under 35 U.S.C. §119(e) of U.S. Patent Application No. 61/193,250 filed 10 Nov. 2008; and U.S. Patent Application No. 61/193,975 filed 14 Jan. 2009, both of which are hereby incorporated herein by reference in their entireties.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA09/01648 | 11/10/2009 | WO | 00 | 7/20/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61193250 | Nov 2008 | US | |
| 61193975 | Jan 2009 | US |
