Nucleic acid purification
10464961 ยท 2019-11-05
Assignee
Inventors
Cpc classification
International classification
C12N15/10
CHEMISTRY; METALLURGY
Abstract
Methods and composition for nucleic acid isolation are provided. In one embodiment, the invention provides a method for nucleic acid purification from biological samples extracted with phenol-based denaturing solvents, which does not require phase separation or nucleic acid precipitation. Methods according to the invention may also be used for differential isolation of RNA and DNA.
Claims
1. A composition at pH of 3.5 to 6.0 and comprising (i) an aqueous component; (ii) a cell lysate as a source of RNA molecules; (iii) guanidinium thiocyanate with a concentration of at least 0.68 M with respect to the composition; (iv) an organic component, wherein the organic component comprises at least 10% phenol by volume; and (v) a silica substrate, said substrate having RNA molecules bound thereto, wherein the composition: (a) does not comprise separate aqueous and organic phases; and (b) is at least 90% free of precipitated RNA molecules.
2. The composition of claim 1, wherein at least 80% of the RNA from the cell lysate is bound to the silica substrate.
3. The composition of claim 1, further comprising a pH buffer.
4. The composition of claim 1, wherein the composition has a pH of less than 4.0.
5. The composition of claim 1, wherein the silica substrate is in the form of beads, fibers or a porous matrix.
6. The composition of claim 1, further comprising an alcohol.
7. The composition of claim 6, wherein the alcohol is a lower alcohol.
8. The composition of claim 1, comprising at least 30% phenol by volume.
9. The composition of claim 1, further comprising a detergent.
10. A method of for purifying RNA molecules from a high phenol solution comprising: (a) obtaining a high phenol monophasic solution at pH 3.5 to 6.0 and comprising (i) an aqueous component; (ii) a cell sample including RNA molecules; (iii) guanidinium thiocyanate with a concentration of at least 0.68 M with respect to the composition; and (iv) an organic component, wherein the monophasic solution comprises at least 10% phenol by volume; (b) contacting the monophasic solution with a silica substrate, thereby binding RNA molecules to the silica substrate, wherein during said contacting the monophasic solution does not comprise separate aqueous and organic phases and wherein the RNA molecules are at least 90% unprecipitated; (c) washing the silica substrate comprising bound RNA molecules with a wash buffer; and (d) eluting the RNA molecules from the silica substrate.
11. The method of claim 10, wherein the eluted RNA molecules are sufficiently purified for sequencing without additional extraction with organic compounds or RNA precipitation steps.
12. The composition of claim 1, comprising at least 20% phenol by volume.
Description
BRIEF DESCRIPTION OF THE DRAWING
(1) The following drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein.
(2)
(3)
(4)
DETAILED DESCRIPTION
(5) The instant invention provides an efficient method for nucleic acid isolation from samples. In particular, methods of the invention allow the binding of nucleic acids directly from an organic sample suspension to a silica substrate. For example, by adding an aqueous binding buffer to an organic suspension DNA can be efficiently bound to a silica matrix and purified away from contaminating protein and cell debris. Likewise, an alcohol based binding buffer can be used to directly bind RNA (and DNA) to silica from an organic suspension. Thus, the methods of the instant invention can be used to purify RNA, DNA, DNA and RNA or to preferentially purify RNA and/or DNA
(6) The direct binding of nucleic acids from an organic suspension offers a number of advantages relative to other protocols for purification. First, the organic and aqueous phases of a suspension do not need to be separated, which is time consuming and typically involves centrifugation of the suspension and laborious removal of the aqueous (or organic) phase. Moreover, because any residual phenol contamination can inhibit the effectiveness of downstream sample treatments (e.g., nucleic acid sequencing), one or more additional step involving extracting the sample with a further organic compound is often required to reduce phenol contamination. Second, there is no need to precipitate nucleic acid out of solution. Again, this process is labor intensive and, in the case of samples containing small amounts of nucleic acid, can result in almost complete loss of the sample's nucleic acid. The direct binding of nucleic acids also has the advantage of reducing the opportunity for nuclease attack of the nucleic acids from the sample. In particular, because the methods of the invention do not involve additional precipitation and centrifugation steps in aqueous buffers the period over which the nucleic acids could be exposed to any active contaminating nuclease is reduced. Likewise, because the sample does not need to be transferred to multiple containers the chance of importing exogenous nuclease is reduced. The foregoing advantages of the new purification methods make them ideal for modern high throughput protocols which require reduced labor input.
(7) I. General Protocol
(8) An illustrative and non-limiting protocol for nucleic acid purification according to the invention is exemplified below.
(9) A. Sample Extraction
(10) Samples may be processed for example, by freeze-thaw, proteinase treatment or mechanical homogenization prior to organic extraction. Organic extraction may be accomplished with a protein denaturing reagent such as a phenol composition or acidic phenol (carbolic acid) composition with guanidinium thiocyanate, to form a suspension. Protein denaturing reagents may additionally comprise components such as a phase separating agent (e.g., chloroform), an antioxidant (e.g., 2-mercaptoethanol or lipoic acid) or a chelating agent. An example composition can comprise phenol:guanidinium thiocyanate:other component(s) (at a ratio of 5:3:2). Once the denaturing solvent is added, the sample may be further homogenized, for example, by vigorous shaking or blending to solubilize all possible cell components. An organic and aqueous phase may form in a sample which comprises phenol, but such phase separation is not required for purification.
(11) B. Addition of Binding Agent
(12) A binding agent is added to the sample. In particular the binding agent comprises a chaotropic salt, an alcohol or a mixture thereof that facilitates nucleic acid binding to a silica substrate. In the case of DNA, the binding agent is a substantially aqueous agent with chaotropic salt(s). A binding agent may also comprise additional buffer agents, salts, detergents and/or alcohol (e.g., lower alcohols such as ethanol or isopropanol). For example, the binding agent may comprise 4-5 M guanidinium thiocyanate (GTC), 5-20% isopropanol, 2-10% glycerol and detergents. In another example, the binding agent comprises 4-5 M GTC, 0.1-1.0 M sodium acetate and, optionally, detergents. One to six volumes of the binding agent are typically added to one volume of a phenol containing sample (e.g., 1:1, 1:2, 1:3, 1:4, 1:5 or 1:6 volumes of binding agent:sample+phenol). In the case of RNA (or RNA/DNA), the binding agent is a substantially alcohol agent, such as ethanol or isopropanol. After addition of a substantially alcohol agent the total concentration of alcohol in the sample is typically raised to greater than about 20%. For embodiments where binding of both DNA and RNA is desired a binding buffer may comprise both a chaotropic salt, such as GTC, and an alcohol.
(13) C. Nucleic Acid Binding to Silica
(14) The sample comprising the binding agent is then contacted to a silica substrate. If an aqueous phase has formed in the sample then the aqueous phase alone may be contacted with the silica substrate. However, separation of organic and aqueous phases is not required for binding. The term silica as used herein refers materials comprising a build-up of silicon and oxygen. Such materials include, without limitation, silica, silicon dioxide, silica gel, fumed silica gel, diatomaceous earth, celite, talc, quartz, glass, glass particles including all different shapes of these materials. Particles, for example, may comprise particles of crystalline silica, soda-lime glasses, borosilicate glasses, and fibrous, non-woven glass. In certain aspects, a silica bead may be a magnetic silica bead (see, e.g., PCT Patent Publn. WO 98/31840, incorporated herein by reference).
(15) Following binding of the nucleic acid the remaining sample suspension is removed by, for example, pipette, vacuum or centrifugation.
(16) D. Optional Addition of a Second Binding Agent
(17) In certain cases, DNA can be bound to silica, while the majority of RNA remains unbound in the sample. In this case, the remaining sample can be collected and a second binding agent added. The binding agent can be a substantially alcohol agent for binding RNA such that, after addition, the total concentration of alcohol in the sample is raised to greater than about 20%, such as to a concentration of about 50% or higher.
(18) E. Optional Second Binding to Silica
(19) The sample and binding agent are contacted with a second silica substrate (as above) to bind additional remaining nucleic acid, such as RNA, from the sample. The remaining sample suspension is then removed as in step 3. The remaining sample may be held for further analysis, such as protein or lipid analysis, or discarded.
(20) F. Wash of Silica Substrate with Bound Nucleic Acid
(21) The silica substrate (and/or second silica substrate) comprising bound nucleic acid is washed one or more times with a washing agent. Washing agents may comprise, for example, solutions comprising alcohol, salts, buffering agents and/or detergents, that do not elute substantial amounts of nucleic acid from the silica. For example, a wash buffer may comprise about or greater than about 40%, 50%, 60%, 70%, 80% or 90% alcohol. In certain aspects the alcohol is a volatile alcohol, such as ethanol. Example washing agents include, but are not limited to, a solution of 20%-50% ethanol and 20%-50% isopropanol; a solution of about 0.1-4.0 M guanidinium hydrochloride, detergents and up to about 80% ethanol; or a solution of about 80% ethanol.
(22) G. Elute Nucleic Acid from the Silica Substrate
(23) The silica substrate (and/or second silica substrate) comprising bound nucleic acids is contacted with an elution buffer to remove the bound nucleic acid into solution. Elution buffers typically comprise a pH buffer agent, limited levels of salts and/or chelating agents. Buffer may additionally comprise nuclease inhibitors.
(24) Following elution nucleic acids may either be further purified or used directly in down-stream analysis such as hybridization or sequencing.
(25) II. Reagents And Kits
(26) Kits may comprise suitably aliquoted reagents of the present invention, such as an acid-phenol denaturing solvent, one or more binding agent and a silica substrate. Additional components that may be included in a kit according to the invention include, but are not limited to, one or more wash buffer, an elution buffer, a proteinase composition, DNase and/or RNase inhibitors, DNase or RNase enzymes, oligonucleotide primers, reference samples (e.g., samples comprising known amounts of DNA or RNA), distilled water, DEPC-treated water, probes, sample vials, polymerase, and instructions for nucleic acid purification. In certain further aspects, additional reagents for DNA and/or RNA clean-up may be included.
(27) The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits of the present invention also will typically include a means for containing reagent containers in close confinement for commercial sale. Such containers may include cardboard containers or injection or blow-molded plastic containers into which the desired vials are retained.
(28) When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being preferred. However, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
(29) III. Examples
(30) The following examples are included to demonstrate certain embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Example 1
RNA Isolation from Human Cells
(31) Various techniques were used for purification of RNA from human cells that were frozen at 80 C. RNA was isolated from 100,000 frozen human cells resuspended in 50 l of water. Samples in lanes 2-6 were lysed in 125 l of RNAzol and further were added one or more of the following, water, ethanol, isopropanol (indicated as Treatment in the Table 1). Samples in lanes 7-10 were lysed in 200 l TRIreagent and further were added one or more of the following, chloroform, ethanol, isopropanol (indicated as Treatment in the Table 1.). Samples in lanes 2-5 and 7-9 were processed by column purification using a silica matrix. Each suspension mixture including all components as indicated in Treatment (Table 1) was loaded onto the Zymo-Spin IC column (Zymo Research, Irvine Calif.), centrifuged at 12,000g, and flow-through discarded. The column was washed once with 700 l RNA Wash Buffer then centrifuged in an empty collection tube to remove residual ethanol. RNA was eluted from the column with 15 l water. Samples 6 and 10 are controls and were processed according to manufacturer's instructions, i.e., to separate aqueous and organic phase, samples were centrifuged. The aqueous phase was then precipitated with isopropanol, RNA pellet washed twice with 70% ethanol, then resuspended in 15 l water. Sample in lane 11 was lysed in 150 l of ZR RNA Buffer and processed according to the protocol for Quick-RNA MicroPrep (Catalog number R1050, Zymo Research, Irvine Calif.).
(32) Any RNA eluted after the indicated treatments was resolved by electrophoresis on a 1% agarose gel. Results from this study are shown in
(33) TABLE-US-00001 TABLE 1 Sample treatments for RNA shown in FIG. 2 Lane Label Treatment 1 1kb 1kb DNA marker 2 RNAzol + H.sub.2O + +water (70 l) + isopropanol (245 l) isopropanol 3 RNAzol + +isopropanol (175 l) isopropanol 4 RNAzol + H.sub.2O + +water (70 l) + 95% ethanol (245 l) ethanol 5 RNAzol + ethanol +95% ethanol (175 l) 6 RNAzol/control +water (70 l) 7 TRIreagent + +isopropanol (250 l) isopropanol 8 TRIreagent + +chloroform (50 l) + 95% ethanol chloroform + ethanol (300 l) 9 TRIreagent + ethanol +95% ethanol (250 l) 10 TRIreagent/control +chloroform (50 l) + isopropanol 11 Quick-RNA - (100 l) control
Example 2
DNA and RNA Isolation from Human Cells
(34) Various techniques were used for purification of nucleic acid molecules from human cells that were frozen at 80 C. Frozen cell samples from 50,000 human cells were resuspended in 25 l of water. All samples (lanes 2-9 and 11) were lysed in 100 l of TriReagent. Following the lysis, a binding agent was added to the sample as indicated under Treatment (Table 2). The mixtures including all components as indicated in Treatment (Table 2) were loaded onto the Zymo-Spin IC column (Zymo Research, Irvine Calif.), centrifuged at 12,000g, and flow-through discarded (sample/lane 2-3, 5, 7-9, 11) or saved for further processing (sample/lane 4 and 6). 95% ethanol was added to the flow-through from samples/lanes 4 and 6 and the mixture was loaded onto the a new Zymo-Spin IC column, centrifuged at 12,000g, and flow-through discarded. After binding, all columns were washed once with 700 l RNA Wash Buffer then centrifuged in an empty collection tube to remove residual ethanol. RNA, DNA or DNA and RNA was eluted from the column with 15 l water. Buffer used in the experiments are as follows: Buffer A (A; 4-5 M GTC, 5-20% isopropanol, 2-10% glycerol and detergents); Buffer B (B; 4-5 M GTC and 0.1 to 1.0 M sodium acetate); Buffer C (C; 4-5 M GTC, 0.1 to 1.0 M sodium acetate and detergents); and Buffer D (D; 1-5M GTC, 1-7M NaI, 20-60% ethanol and detergent).
(35) Any nucleic acid that was eluted from the column was resolved by electrophoresis on a 1% agarose gel. Results from this study are shown in
(36) TABLE-US-00002 TABLE 2 Sample treatments for results shown in FIG. 3 Lane Label Treatment 1 1kb 1kb DNA marker 2 D +Buffer D (375 l) 3 A +Buffer A (375 l) 4 B +Buffer B (375 l) 5 B/FT Sample/lane 4 column flow-through + 95% ethanol (500 l) 6 C +Buffer C (375 l) 7 C/FT Sample/lane 6 column flow-through + 95% ethanol (500 l) 8 Ethanol +95% ethanol (125 l) 9 Isopropanol +Isopropanol (125 l) 10 1kb 1 kb DNA marker 11 B + Ethanol +Buffer B (250 l) + 95% ethanol (375 l)
REFERENCES
(37) Each of the foregoing documents is hereby incorporated by reference in its entirety:
(38) U.S. Pat. Nos. 4,843,155; 5,472,872; 6,210,945
(39) U.S. Patent Publication 20100222560
(40) PCT Patent Publn. WO 98/31840.
(41) Sambrook et al., In: Molecular Cloning-A Laboratory Manual, 2001.