METHOD FOR EXTRACTION OF CELL-FREE DNA
20220403372 · 2022-12-22
Assignee
Inventors
Cpc classification
C12N15/1006
CHEMISTRY; METALLURGY
International classification
Abstract
Provided herein are compositions and methods for extracting cell-free DNA (cfDNA) from a biological sample without the need for pretreatment with proteases prior to binding of the cfDNA to a solid substrate and without a heated elution step. The methods comprise contacting the biological sample with a chelating agent and an oxidizing agent prior to eluting the cell-free DNA from a solid substrate.
Claims
1. A method for extracting cell-free DNA (cfDNA) from a biological sample, the method comprising: (1) combining the biological sample with a solid substrate and a binding buffer comprising a chaotropic agent in order to bind cfDNA from the biological sample to the solid substrate, wherein the biological sample has not been treated with proteinase K; (2) washing the cfDNA bound to the solid substrate with a wash buffer; and (3) eluting the cfDNA from the solid substrate, wherein the binding buffer and/or the wash buffer comprises a chelating agent and an oxidizing agent.
2. The method of claim 1, wherein the binding buffer comprises a chaotropic agent, a chelating agent, and an oxidizing agent.
3. The method of claim 1, wherein the wash buffer comprises a chelating agent and an oxidizing agent.
4. The method of claim 1, wherein eluting is performed at ambient temperature.
5. The method of claim 1, wherein the method does not include a heat treatment step.
6. The method of claim 1, wherein the chelating agent is an amine-based chelating agent or a phosphate-based chelating agent.
7. The method of claim 1, wherein the oxidizing agent is sodium percarbonate or hydrogen peroxide.
8. The method of claim 1, wherein the chelating agent is EDTA or MGDA and the oxidizing agent is sodium percarbonate.
9. The method of claim 1, wherein the chelating agent is STP and the oxidizing agent is sodium percarbonate or hydrogen peroxide.
10. The method of claim 1, wherein the binding buffer comprises an alcohol.
11. The method of claim 1, wherein the binding buffer comprises isopropyl alcohol.
12. The method of claim 1, wherein the solid substrate is a silica substrate.
13. The method of claim 1, wherein the solid substrate is silica beads, silica-coated beads, a silica column, or a silica membrane.
14. The method of claim 1, wherein the biological sample is a blood plasma sample.
15-33. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] The following drawings form 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 one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0029]
[0030]
[0031]
[0032]
[0033]
DETAILED DESCRIPTION
[0034] One commonly used method for extraction of nucleic acids from biological samples involves the addition of a chaotropic agent to a complex biological sample to form a lysate, followed by binding of nucleic acids to a solid silica substrate (U.S. Pat. No. 5,234,809). Under some conditions, the addition of alcohol to the lysate may improve nucleic acid binding to the silica substrate. After binding of the nucleic acids to the silica substrate and removal of the non-bound components, one or more washes are performed, followed by elution of the nucleic acids from the substrate. The silica substrate may be provided in the form of a packed column (stationary solid phase) or in the form of particles (mobile solid phase), which may have magnetic or paramagnetic cores to facilitate separation from the non-binding components of the lysate.
[0035] Efficient isolation of nucleic acids from blood products using the aforementioned method is known to be challenging. In particular, blood products tend to plug stationary solid phase systems, preventing the lysate from passing through the solid phase. Use of blood products with mobile solid phase systems generally result in poor yields of nucleic acids. To overcome this problem, blood product lysates are typically treated with a proteinase K to remove unwanted protein prior to addition to silica substrates. However, such treatments add to the cost and complexity of nucleic acid extraction, as they often require prolonged heat treatment and separate addition of proteinase K and chaotropic agents and alcohol.
[0036] In recent years, interest in analysis of circulating or cell-free DNA (cfDNA) has grown. By way of example, cfDNA can be analyzed for the diagnosis and prognosis, as well as the early detection, of cancer; pre-natal diagnosis of fetal genetic conditions; detection of biomarkers of myocardial infarction or transplant graft rejection. cfDNA is extracted from blood plasma, and various methods have been developed to optimize extraction of these extracellular nucleic acids (see, e.g., Jorgez et al., Genet. Med. (2006) 8:615-619; U.S. Pat. Publn. US2017/0183712; U.S. Pat. Publn. US2019/0225958). cfDNA exists within nucleosomes and efficient extraction for subsequent analysis is dependent on releasing the nucleic acids from the protein components of the nucleosomes. One well established commercially available method makes use of the QIAamp® Circulating Nucleic Acid Kit (Qiagen N.V.), which includes a pretreatment with proteinase K prior to addition of the lysate to the solid silica matrix.
[0037] The compositions and methods disclosed herein eliminate the need for pretreatment with proteases prior to binding of nucleic acids to silica substrates. In one embodiment, plasma containing nucleic acids of interest is added to a solution containing a high concentration of a chaotropic agent in the presence of a chelating agent (e.g., EDTA, MGDA, or sodium triphosphate) and sodium percarbonate. In another embodiment, nucleic acids bound to a solid substrate are washed with a solution containing a chelating agent (e.g., EDTA, MGDA, or sodium triphosphate) and sodium percarbonate. In alternate embodiments, sodium percarbonate is replaced by hydrogen peroxide in the presence of sodium triphosphate. By eliminating the requirement for pretreating the biological samples with proteinase K prior to binding nucleic acids to the silica substrate, the complexity, cost, and time taken to extract nucleic acids for further analysis is reduced. The disclosed composition and methods also facilitate elution without the need for heat as well as the addition of alcohol (e.g., isopropyl alcohol) to the binding buffer prior to use in the extraction with equivalent results.
I. DEFINITIONS
[0038] As used herein the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising,” the words “a” or “an” may mean one or more than one.
[0039] The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” As used herein “another” may mean at least a second or more.
[0040] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the inherent variation in the method being employed to determine the value, the variation that exists among the study subjects, or a value that is within 10% of a stated value.
[0041] As used herein, “essentially free,” in terms of a specified component, is used herein to mean that none of the specified component has been purposefully formulated into a composition and/or is present only as a contaminant or in trace amounts. The total amount of the specified component resulting from any unintended contamination of a composition is therefore well below 0.05%, preferably below 0.01%. Most preferred is a composition in which no amount of the specified component can be detected with standard analytical methods.
[0042] By “biological sample” is meant a sample comprising any biological material from which samples can be prepared for use in the methods disclosed herein. This includes, but is not limited to, blood, plasma, serum, saliva, urine, cerebral spinal fluid, pleural fluid, milk, lymph, sputum, and semen.
[0043] By “chelator” or “chelating agent” is meant chemical compounds that react with metal ions to form a stable, water-soluble complex. The chelator may be an amine-based chelator or a phosphate-based chelator. The chelator may be ethylenediaminetetraacetic acid (“EDTA”), ethylene glycol-bis(2-aminoethylether) tetraacetic acid (“EGTA”), methylglycinediacetic acid (“MGDA”), sodium triphosphate (“STP”), nitrilotriacetic acid (“NTA”), iminodisuccinic acid (“IDS”), polyaspartic acid, S,S-ethylenediamine-N,N′-disuccinic acid (“EDDS”), L-Glutamic acid N,N-diacetic acid, tetrasodium salt (“GLDA”), or their salts. The terms “EDTA,” “MGDA,” etc. will be used to refer both to the acid and the salt form, and either form may be used in the present invention.
[0044] By “oxidizing agent” is meant a substance that accepts electrons from another substance and is thus itself reduced. Exemplary oxidizing agents include peroxide-containing compounds, such as, for example, hydrogen peroxide, sodium percarbonate, urea peroxide, melamine peroxide, potassium percarbonate, sodium perborate, potassium perborate, sodium peroxide, potassium peroxide, magnesium peroxide, barium peroxide, calcium peroxide, strontium peroxide, hydrogen peroxide adducts of sulfates, hydrogen peroxide adducts of phosphates, hydrogen peroxide adducts of pyrophosphates, and the like
[0045] By “chaotropic agent” is meant agents that disrupt molecular structure, particularly molecular structure formed by nonbonding forces such as hydrogen bonding, Van der Waals interaction, and hydrophobic effect. Chaotropic agents are well known in the field of biochemistry and include, but are not limited to, guanidine hydrochloride, guanidine thiocyanate, guanidine isothiocyanate, sodium perchlorate, sodium iodide, sodium trichloroacetate, urea, thiourea, rhodanite salt, aminoguanidine bicarbonate, guanidine carbonate, guanidine phosphate, and aminoguanidine hydrochloride.
[0046] By “ambient temperature” is meant room temperature and generally refers to a temperature that is between about 20° C. and about 30° C. (e.g., about 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., or 30° C.).
[0047] As used herein “nucleic acid” means either DNA or RNA, either single-stranded or double-stranded.
[0048] As used herein, “amplification” or “amplifying” refers to the in vitro production of additional copies of a target nucleic acid sequence. Amplification is generally carried out using polymerase chain reaction (PCR) technologies known in the art. The term “amplification reaction” refers to an aqueous solution comprising the various reagents used to amplify a target nucleic acid. These may include enzymes (e.g., a thermostable polymerase), aqueous buffers, salts, amplification primers, target nucleic acid, nucleoside triphosphates, and optionally, at least one labeled probe and/or optionally, at least one agent for determining the melting temperature of an amplified target nucleic acid (e.g., a fluorescent intercalating agent that exhibits a change in fluorescence in the presence of double-stranded nucleic acid).
[0049] The term “PCR” or “polymerase chain reaction” encompasses derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, assembly PCR, digital PCR, and the like. “Real-time PCR” means a PCR for which the amount of reaction product, i.e., amplicon, is monitored as the reaction proceeds. There are many forms of real-time PCR that differ mainly in the detection chemistries used for monitoring the reaction product, e.g., U.S. Pat. No. 5,210,015 (“Taqman”); U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes); U.S. Pat. No. 5,925,517 (molecular beacons). Detection chemistries for real-time PCR are reviewed in Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002). “Nested PCR” means a two-stage PCR wherein the amplicon of a first PCR becomes the sample for a second PCR using a new set of primers, at least one of which binds to an interior location of the first amplicon. “Initial primers” in reference to a nested amplification reaction mean the primers used to generate a first amplicon, and “secondary primers” mean the one or more primers used to generate a second, or nested, amplicon. “Multiplexed PCR” means a PCR wherein multiple target sequences (or a single target sequence and one or more reference sequences) are simultaneously carried out in the same reaction mixture. Usually, distinct sets of primers are employed for each sequence being amplified. “Quantitative PCR” means a PCR designed to measure the abundance of one or more specific target sequences in a sample or specimen. “Digital PCR” involves partitioning the sample such that individual nucleic acid molecules contained in the sample are localized in many separate regions, such as in individual wells in microwell plates, in the dispersed phase of an emulsion, or arrays of nucleic acid binding surfaces. Each partition will contain 0 or 1 molecule, providing a negative or positive reaction, respectively.
II. EXAMPLES
[0050] The following examples are included to demonstrate preferred 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 inventor 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 spirit and scope of the invention.
Example 1—Plasma Preparation and Buffer Preparation
[0051] In order to prepare plasma, blood was drawn into EDTA-containing tubes and centrifuged at 1900 g for 10 min to sediment cellular material. The supernatant containing the plasma was collected and recentrifuged at 6000 g for 10 min. Then, the supernatant plasma was collected and stored at −80° C. until needed.
[0052] Modified Binding Buffer 1 (MBB1) was prepared as follows: 3.4 M guanidine, 37 mM TRIS, 2.5% Tween 20, 2.3% Triton X-100, 3.8% Simethicone Emulsion, 20 mM EDTA, 40 mM sodium percarbonate, 22% isopropyl alcohol (IPA). The solution was stored for future use at ambient temperature protected from light.
[0053] Modified Binding Buffer 2 (MBB2) was prepared as follows: 3.4 M guanidine, 37 mM TRIS, 2.5% Tween 20, 2.3% Triton X-100, 3.8% Simethicone Emulsion, 11 mM MGDA (methylglycinediacetic acid trisodium salt), 40 mM sodium percarbonate, 22% isopropyl alcohol (IPA). The solution was stored for future use at ambient temperature protected from light.
[0054] Modified Binding Buffer 3 (MBB3) was prepared as follows: 3.4 M guanidine, 37 mM TRIS, 2.5% Tween 20, 2.3% Triton X-100, 3.8% Simethicone Emulsion, 14 mM STP (sodium triphosphate), 40 mM sodium percarbonate, 22% isopropyl alcohol (IPA). The solution was stored for future use at ambient temperature protected from light.
[0055] Modified Binding Buffer 4 (MBB4) was prepared as follows: 3.4 M guanidine, 37 mM TRIS, 2.5% Tween 20, 2.3% Triton X-100, 3.8% Simethicone Emulsion, 14 mM STP (sodium triphosphate), 5 mM hydrogen peroxide, 22% isopropyl alcohol (IPA). The solution was stored for future use at ambient temperature protected from light.
Example 2—cfDNA Extraction Using EDTA or MGDA and Sodium Percarbonate
[0056] Cell-free DNA (cfDNA) was extracted from human plasma using methods disclosed herein, and the extracted cfDNA was used for further analysis by PCR. Results were compared to those obtained using standard methods of extraction with and without pretreatment using proteinase K and to those obtained using a commercially available kit for cfDNA extraction (QIAamp®, obtained from Qiagen N.V.).
[0057] Extraction using the standard mobile solid phase (Standard) method was performed as follows. First, 1 mL of human plasma was obtained as described in Example 1, added to 500 μL proteinase K (QIAGEN; Cat. No. 19131), and incubated at 60° C. for 30 min. Then, 2.5 mL of standard binding buffer (4.5 M guanidine, 50 mM TRIS, 3.3% Tween 20, 3% Triton X-100, 5% Simethicone Emulsion), 750 μL isopropyl alcohol (IPA), and 250 μL magnetic silica particles (Luminex Corp.) were added to the incubated plasma, and then the mixture was vortexed. The mixture was incubated at ambient temperature for at least 10 min, and the particles were collected on the side of the tube to permit removal of the liquid. Particles were washed once with Wash Buffer 1 (0.5 M guanidine, 2.5% Triton X-100, 2.5% Tween 20, 50% IPA, 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), 2 mM EDTA) and once with Wash Buffer 2 (pH 5.5, 20 mM MES). Nucleic acids were eluted from the particles in 50 μL, Elution Buffer (10 mM Tris, pH 8.1) at 70° C. for 10 min on an Eppendorf ThermoMixer® at 1000 rpm.
[0058] Extraction using the Standard method without pretreatment with proteinase K was performed by omitting the addition of proteinase K and the 60° C. incubation.
[0059] Extraction using the QIAamp® kit was performed in accordance with the manufacturer's instructions.
[0060] Extraction using the disclosed MBB1 and MBB2 methods was performed as follows. First, 1 mL of human plasma was obtained as described in Example 1 and added to 3.250 mL Modified Binding Buffer 1 (MBB1) or Modified Binding Buffer 2 (MBB2). After briefly vortexing, 100 μL, of magnetic silica particles, as described above, were added to the mixture, which was vortexed to resuspend the particles. After a 7-minute incubation at ambient temperature, particles were collected on the side of the tube to permit removal of the liquid. Particles were washed once with Wash Buffer 1 (0.5 M guanidine, 2.5% Triton X-100, 2.5% Tween 20, 50% IPA, 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), 2 mM EDTA) and once with Wash Buffer 2 (pH 5.5, 20 mM MES). Nucleic acids were eluted from the particles by vortexing to resuspend the magnetic particles in 50 μL, Elution Buffer (10 mM Tris, pH 8.1) and incubating at ambient temperature for 30 sec.
[0061] PCR analysis of the extracted nucleic acids was performed as follows. A TAQMAN® assay using the RPP30 human reference gene as a target was used to evaluate suitability of extracted nucleic acid for subsequent analysis. C.sub.t values from qPCR of nucleic acids extracted using the various methods were compared. Eluted nucleic acid (5 μL) was added to 25 μL, PCR Master Mix (PHOENIX™ Taq DNA Polymerase (QIAGEN) 0.08 U/μL; 10 mM Bis-Tris propane, 90 nM DTT, 6 mg/mL BSA, 10 mM dTTP, 10 mM dGTP, 10 mM dCTP, 10 mM dATP, 10 nM Tris, 2.5 nM MgCl.sub.2, 50 nM KCl). Primers and probe were added (200 nM Forward: 5′-GATTTGGACCTGCGAGCG-3′; 200 nM Reverse: 5′-GCGGCTGTCTCCACAAGT-3′; 400 nM Probe: 5′-VIC-CTGACCTGAAGGCTCT-3′). PCR reactions were cycled on an ABI 7500 instrument as follows: 140 sec at 95° C., followed by 45 cycles of 10 sec at 95° C. and 30 sec at 58° C. Fluorescence was measured in the VIC channel set up for NFQ-MGB quench.
[0062] cfDNA extracted using the disclosed MBB1 and MBB2 methods yielded similar C.sub.t values to the Standard method with proteinase K treatment or the kit (
Example 3—cfDNA Extraction Using Sodium Triphosphate and Sodium Percarbonate or Hydrogen Peroxide
[0063] cfDNA was extracted from human plasma using the MBB1 and MBB2 methods described in Example 2, except that Modified Binding Buffer 3 (MBB3) and Modified Binding Buffer 4 (MBB4) were used. Results were compared to those obtained using the Standard method, the Standard method without pretreatment with proteinase K, and the MBB1 method, each described in Example 2.
[0064] Note that 200 μL of plasma was used in these experiments and other reagents were adjusted accordingly to maintain the same relative concentrations as described in Example 2. The results are shown in
Example 4—cfDNA Extraction Using Either a Chelating Agent or Sodium Percarbonate
[0065] Aliquots of plasma (200 μL) were processed (a) using the Standard method without pretreatment with proteinase K, as described in Example 2, (b) using the QIAamp® kit in accordance with the manufacturer's instructions, (c) using the Standard method without pretreatment with proteinase K, except that the standard binding buffer contained 15, 35, or 75 mM MGDA, and (d) using the Standard method without pretreatment with proteinase K, except that the standard binding buffer contained 25, 65, or 125 mM sodium percarbonate. Results are shown in
Example 5—Addition of a Chelating Agent and Sodium Percarbonate in a Commercial Kit
[0066] cfDNA was extracted from human plasma using the QIAamp® cfDNA kit protocol. One sample was processed according to the manufacturer's protocol. A second sample was processed according to the same protocol, but with the proteinase K step omitted. A third sample was also processed without the proteinase K step, but with the buffer provided in the kit modified to include a chelating agent (20 mM EDTA) and sodium percarbonate (40 mM).
[0067] Omitting the proteinase K step resulted in no amplifiable nucleic acid being recovered (
Example 6—Extraction of cfDNA Using Modified Wash Buffer
[0068] Nucleic acid was extracted from 200 μL aliquots of plasma collected and prepared as described in Example 1. A first aliquot was processed using the Standard method with proteinase K pretreatment as described in Example 2. A second aliquot was processed using the Standard method without proteinase K pretreatment as described in Example 2. A third aliquot was processed using the Standard method without proteinase K pretreatment, but with Wash Buffer 1 modified to include chelating agent (25 mM EDTA) and sodium percarbonate (50 mM). Note that for all three aliquots, remaining reagent volumes as provided in Example 2 were adjusted to account for the reduced sample volume.
[0069] The addition of chelating agent and sodium percarbonate to the Wash Buffer 1 significantly improved the efficiency of extraction of cfDNA when compared to the Standard method without a proteinase K pretreatment step (
[0070] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein 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.
REFERENCES
[0071] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
[0072] U.S. Pat. No. 5,210,015
[0073] U.S. Pat. No. 5,234,809
[0074] U.S. Pat. No. 5,925,517
[0075] U.S. Pat. No. 6,174,670
[0076] U.S. Pat. No. 6,569,627
[0077] U.S. Pat. Publn. US2017/0183712
[0078] U.S. Pat. Publn. US2019/0225958
[0079] Jorgez et al., Genet. Med., 8:615-619 (2006)
[0080] Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002)