Patent Application
20080003585
The FIGURE depicts a microfluidics device of the present invention.
Diatomaceous earth (DE), also known as kieselguhr or diatomite, is a loosely coherent chalk-like sedimentary rock consisting primarily of fragments and shells of hydrous silica secreted by diatoms, which are microscopic one-celled algae. The primary component of DE is silica and it is highly porous in structure. DE is commercially available in natural form as well as in calcined and flux-calcined forms. Calcined DE is DE that has been calcined at temperatures in the vicinity of 900-1,000° C., while flux-calcined DE is DE that has been calcined in the presence of soda ash or sodium chloride to reduce the surface area. The various forms of DE are sold under such tradenames as CELITE® (registered trademark of JohnsManville Corp., Lompoc, Calif., USA) and CELATOM® (registered trademark of EaglePicher Filtration and Minerals, Inc., Reno, Nev., USA).
The diatomaceous earth used in the practice of this invention can be in particulate form, or immobilized in a membrane, or packed in porous retainer such as a flow-through cartridge. When in particulate form, the particle size can vary and is not critical to the invention. In preferred embodiments of the invention, the particles are from about 1 μm to about 125 μm in diameter, most preferably from about 5 μm to about 60 μm in diameter.
In procedures in which the first step involves binding all nucleic acids in the lysate to the diatomaceous earth, certain operating conditions will promote the binding, depending on the particular cell lysate and the form of the diatomaceous earth. In certain embodiments of the invention, these conditions include the presence of a chaotropic agent in the solid-liquid medium. Examples of suitable chaotropic agents are guanidinium thiocyanate, guanidinium isothiocyanate, guanidinium hydrochloride, alkali iodides such as sodium or potassium iodide, and alkali perchlorates such as sodium or potassium perchlorate. Guanidinium thiocyanate is preferred. The concentration of the chaotropic agent when present may vary and the precise amount is not critical. The benefit resulting from the presence of the chaotropic agent will generally be achieved at a wide range of concentrations of the agent, with best results generally obtained using concentrations ranging from about 0.8 M to about 10 M. Conditions that promote the binding of the nucleic acids to the diatomaceous earth may also include incubating the solid-liquid medium in the presence of a buffer, preferably one that maintains a pH of from about 6.4 to about 9.5. When both a chaotropic agent and a buffer are used, the chaotropic agent can be combined with the buffer in an aqueous solution which is then added to the solid-liquid medium. An example of a chaotropic agent-containing buffer solution that can be used to bind all nucleic acids to the diatomaceous earth is one whose composition is 6.0 M sodium perchlorate, 0.05 M Tris-Cl pH 8, and 10.0 mM ethylenediamine tetraacetic acid.
In procedures in which selective binding is achieved by the use of a special buffer solution, selectivity can be achieved by a buffer solution that promotes the binding of double-stranded nucleic acids in preference to single-stranded nucleic acids. This will be useful in purifying DNA from mixtures in which RNA is present only in single-stranded form. Procedures and an appropriate buffer solution for achieving this effect are disclosed in the Beld et al. paper referenced above. The selective binding buffer is a lysis/binding buffer whose composition is guanidinium thiocyanate in 0.2M ethylenediamine tetraacetic acid at pH 8.0, prepared by dissolving 120 g of the guanidinium thiocyanate in 100 mL of the EDTA. In procedures where RNA is the nucleic acid of interest, the RNA can be isolated from the DNA by the use of a combination of aqueous and organic solvents disclosed in Chomczynski, P., U.S. Pat. Nos. 4,843,155 and 5,346,994. According to these patents, RNA can be isolated from DNA by an aqueous solvent solution containing phenol and a guanidinium compound at a pH of 4, followed by extraction of the aqueous solution with an organic solvent such as chloroform. The RNA remains in the aqueous phase and is precipitated by adding a lower alcohol prior to being bound to the diatomaceous earth for final purification in the microfluidics device.
Preferred conditions promoting the binding of the nucleic acids to diatomaceous earth in any of the embodiments of this invention are a moderate temperature, preferably from about 15° C. to about 30° C., most preferably about 22° C., and a contact time of from about 3 minutes to about 60 minutes, most preferably from about 5 minutes to about 20 minutes. When a lysate is contacted directly with the diatomaceous earth, the volume ratio of the lysate to the diatomaceous earth can likewise vary, although effective results can be achieved with a volume ratio of approximately 1:1.
The wash buffer used to remove unbound species from the diatomaceous earth after the binding of the nucleic acids can be any buffer that maintains an appropriate pH and separates unbound species from the diatomaceous earth. The wash buffer can also contain the chaotropic agent and can either be the same buffer used to promote the binding of the nucleic acids to the diatomaceous earth or a distinct buffer. In certain embodiments of the invention, the wash buffer contains a lower alcohol such as methanol, ethanol or isopropanol. Ethanol is particularly preferred. When an alcohol-containing buffer is used, the alcohol can constitute from about 20% to about 95% of the buffer on a volume basis. The alcohol-containing wash buffer can also include a salt such as sodium chloride. An example of an alcohol-containing wash buffer that does not contain one of the chaotropic agents listed above is one of the following composition: 20.0 mM Tris-Cl pH 7.5, 2.0 mM EDTA, 0.4 M NaCl, and 50% ethanol. In certain embodiments of the invention, washing is achieved by purging the diatomaceous earth first with a binding buffer, i.e., one that contains a chaotropic agent and a buffer as described in the preceding paragraph, and then with an alcohol-containing buffer that does not contain a chaotropic agent, as described in this paragraph. The total volume of buffer used in this washing step is preferably two or more times the volume of the solid-liquid medium as a whole, and when successive buffers are used, the volume of each is preferably two or more times the volume of the solid-liquid medium.
For procedures that utilize the enzymes RNase and DNase, these enzymes are available commercially from suppliers of chemicals for biological laboratories. Examples of such suppliers are Promega Corporation, Madison, Wis., USA, and Qiagen Inc., Valencia, Calif., USA. Information regarding the optimal amounts or concentrations of the enzyme and the inclusion of any necessary buffers or additives are also available from the suppliers. These and other incubation conditions such as temperature and contact time will also be readily apparent to those skilled in the use of these enzymes.
After the incubation with the appropriate enzyme is completed, removal of the cleaved nucleic acid—RNA in cases where DNA is being purified, and DNA in cases where RNA is being purified—is achieved by a second wash, which can be performed using the same wash buffer or sequence of buffers as used prior to the cleavage or a different wash buffer or buffer sequence. Any buffer that removes unbound nucleic acid and causes no structural transformation of the bound nucleic acid can be used. The environment and operating conditions will be the same as those of the first wash buffer.
Elution of the purified nucleic acid in all embodiments of the present invention is achieved by purging the diatomaceous earth with an elution buffer. Any buffer that will dissociate the nucleic acid from the diatomaceous earth and is otherwise inert to the nucleic acid can be used. The preferred buffer is a low salt buffer, i.e., one with a maximum salt concentration of about 20 mM. A preferred pH range of the buffer is about 7.5 to about 8.5. One example of a low salt buffer is an aqueous solution containing 10.0 mM Tris-Cl pH 8 and 1.0 mM EDTA (ethylenediaminetetraacetic acid). Another example is DEPC (diethylpyrocarbonate)-treated water.
An example of a protocol for RNA preparation is as follows. Whole cells are first mixed with a lysis solution consisting of 4M guanidine thiocyanate, 20 mM Tris, 20 mM EDTA, pH 7, supplemented with 1% mercaptoethanol, either in a tube or within the DE-containing well in the microfluidics device that is designated for sample preparation. An equal volume of 70% ethanol is then added and the mixture is thoroughly mixed, then incubated in the sample preparation well for 1-60 minutes. The liquid phase is then discarded into a waste well and the DE is washed with a low-stringency buffer consisting of 50 mM Tris, pH 7.5 (prepared from a 5× concentrate by dilution with 95-100% ethanol). Unbound material is then discarded into the waste well. DNase I, reconstituted from a lyophilized powder in Tris, pH 7.5, then mixed one part with 15 parts of DNase Dilution Buffer (40 mM Tris-HCI pH 8, 10 mM MgSO4, 10 mM CaCl2), is added to the sample preparation well and digestion is allowed to proceed for fifteen minutes at room temperature. The DNase solution is then discarded from the sample preparation well and the DE is first washed with a high-stringency wash buffer consisting of 2.5M guanidine hydrochloride, 10 mM Tris, 10 mM EDTA, pH 7, then the low-stringency wash buffer. The RNA is then eluted with 4-30 μL of DEPC-treated water or T10E1 elution solution (10 mM Tris, 1 mM EDTA, pH 8.0).
A similar protocol although with RNase instead of DNase can be used for DNA preparation, including a lysis/binding buffer that separates double- from single-stranded nucleic acids. A buffer that can be used for the RNase treatment is 10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 0.3M NaCl.
In embodiments of the invention that utilize diatomaceous earth in particulate form, the solid-liquid medium that includes the diatomaceous earth and the cell lysate or any buffer or wash solution being used at the various stages of the purification procedure will be a suspension of the solid diatomaceous earth particles in the liquid. The suspension in these embodiments can be retained in a reservoir within the microfluidics device by a particle-retaining filter. The filter can be any structure that allows the liquid phase of the suspension to pass while blocking the passage of the diatomaceous earth particles. Examples of such filters are frits, porous membranes, and mesh screens. The pore or aperture size in the filter will be small enough to retain the diatomaceous earth particles. For diatomaceous earth with a particle size range of 5 μm to 60 μm, a preferred filter pore diameter is approximately 3 μm.
Microfluidics devices to which the present invention can be applied are generally those of the type described in the references cited above. As shown in the FIGURE attached to this specification, the typical microfluidics device 11 is characterized by a body structure that contains cavities in the form of microchannels 12 that have at least one dimension that is 500 microns or less, and in many cases 100 microns or less. The typical sample reservoir 13 in which the diatomaceous earth 14 is retained is generally larger than the microchannels that supply the reservoir or draw wash liquid or eluate from it, and a filter 15 as described above retains the diatomaceous earth 14 in the reservoir 13. In most cases, the sample reservoir has a volume ranging from about 5 μL to about 50 μL. The conveyance of liquids into and out of the reservoirs by way of the microchannels can be achieved by any conventional techniques, examples of which are electrophoretic transport, pneumatic transport, and hydraulic transport. One example of a transport system is that disclosed in Boronkay, G., et al., U.S. patent application Ser. No. 11/288,838, filed Nov. 28, 2005. Where necessary, liquids can be directed into particular microchannels and the direction of flow can be reversed or re-directed by conventional methods as well. One example of a method for selecting a liquid flow path among two or more alternative flow paths is the use of electrophoretic forces with selective use of electrodes. Another is the use of pneumatic or hydraulic means in conjunction with microfluidic valves. Such valves are known in the microfluidics art and include rotary valves and diaphragm valves such as those disclosed in Hartshorne, H. A., et al., U.S. Pat. No. 6,748,975 B2, issued Jun. 15, 2004, and bubble valves such as those disclosed in Gilbert, J. R., et al., U.S. Pat. No. 6,877,528 B2.
As noted above, the durations of the various steps of the purification process will be varied and chosen to achieve the optimal result for each step, whether the step be one that involves binding to the diatomaceous earth, the action of an enzyme, washing, or elution. The optimal duration for each step will be known to those skilled in the art or readily determinable by routine experimentation. The selected duration can be achieved by selecting the volume of the particular liquid medium that is being conveyed through a reservoir, the volume of the reservoir, the volumetric flow rate through the reservoir and the microchannels supplying the reservoir, and other parameters and operating conditions of the system.
Once the DNA or RNA purification is completed, PCR reaction materials are added, either directly or following the addition of the reverse transcriptase in the case of purified RNA. These additions and the incubations needed for PCR are performed downstream of the purification stages but within the same microfluidics device. The amplification reactions themselves are likewise performed within the device. The amplification reactions can be performed in the same manner as in the prior art, using reaction mixtures and reaction conditions that are used or have been disclosed for use in these reactions. Means of forming the mixtures, initiating the reactions, and performing them within the microfluidics device can thus be the same as disclosed, for example, in the Kopp et al. paper (Science 1998), the Knapp et al. U.S. Patent Application Publication No. US 2005/0042639 A1, and the Wada et al. U.S. Patent Application Publication No. US 2005/0170362 A1, all cited above. Temperature changes can be effected by maintaining different regions of the microfluidics device at different temperatures by the use of thermoelectric modules or other localized temperature control means, and passing the reaction mixture through the different stages in succession, while durations of exposure to the different temperatures can be controlled by of the manufacture of microchannels of selected lengths in the temperature-controlled sections and the flow rate, the length in each section establishing the residence time of the appropriate reaction mixture at the temperature of that section. Alternatively, thermally cycling can be achieved by heating and cooling the entire microfluidics device to the temperatures required for each stage of the amplification procedure.
The foregoing description is offered primarily for purposes of illustration. Further variations, modifications, and substitutions that can be utilized in implementing the novel concepts of the invention and will therefore fall within the scope of the claims will be apparent to those skilled in the art of PCR, microfluidics, and diatomaceous earth.
