WO2014159719A1 - Procédés permettant d'isoler des acides nucléiques - Google Patents

Procédés permettant d'isoler des acides nucléiques Download PDF

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Publication number
WO2014159719A1
WO2014159719A1 PCT/US2014/024900 US2014024900W WO2014159719A1 WO 2014159719 A1 WO2014159719 A1 WO 2014159719A1 US 2014024900 W US2014024900 W US 2014024900W WO 2014159719 A1 WO2014159719 A1 WO 2014159719A1
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WIPO (PCT)
Prior art keywords
hours
magnetic particle
composition
cells
gauss
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PCT/US2014/024900
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English (en)
Inventor
Shawn Patrick GROGAN
Darryl D. D'LIMA
Sungho Jin
Martin Lotz
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Scrips Health
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Publication of WO2014159719A1 publication Critical patent/WO2014159719A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads

Definitions

  • a method of purifying a nucleic acid comprising: i) incubating a magnetic particle with a composition comprising intact cells, ii) disrupting the membranes of the cells; and iii) separating the nucleic acid from the cell debris and contaminants; wherein the cells are of eukaryotic origin.
  • the magnetic particle comprises Fe 3 0 4 .
  • the magnetic particle comprises Fe 2 0 3 .
  • the magnetic particle is added at a concentration of 0.05 to 1.0 mg per 5 x 10 5 cells.
  • the magnetic particle is uncoated.
  • the composition has a non- acidic pH.
  • the composition has a neutral or basic pH. In some embodiments, the composition has a pH of at least about 4.0, 5,0, 6,0 or 7.0. In some embodiments, the composition has a pH of about 4.0. In some embodiments, the composition has a pH of about 7.0. In some embodiments, the composition has a physiological pH. In some embodiments, the cells are of animal, plant, or fungal origin. In some embodiments, the nucleic acid is DNA, RNA or PNA. In some embodiments, the nucleic acid is RNA.
  • the RNA is an RNA selected from mRNA, miRNA, non-coding RNA (ncRNA), tRNA, rRNA, pre-mRNA, tmRNA, piRNA, CRISPR RNAs, snRNA, viral RNA, and dsRNA.
  • the nucleic acid is DNA.
  • the method further comprises immobilizing the magnetic particle with a magnetic field.
  • the magnetic field is provided by a magnet.
  • the magnet is about 10 gauss, about 20 gauss, about 30 gauss, about 40 gauss, about 50 gauss, about 60 gauss, about 70 gauss, about 80 gauss, about 90 gauss, about 100 gauss, about 120 gauss, about 140 gauss, about 160 gauss, about 180 gauss, about 200 gauss, about 300 gauss, about 400 gauss, or about 500 gauss.
  • the magnet is about 100 gauss.
  • the cells are derived from tissue.
  • the cells are derived from blood, plasma, or urine.
  • the cells are human.
  • the cells are in culture. In some embodiments, the cells are adherent cells. In some embodiments, the cells are in suspension. In some embodiments, the cells are in suspension in a buffered solution. In some embodiments, the cell membranes are disrupted chemically. In some embodiments, the cell membranes are disrupted physically.
  • the cells are incubated with the magnetic particle for about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours, about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours, about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours, about 16 hours, about 16.5 hours, about 17 hours, about 17.5 hours, about 18 hours, about 18.5 hours, about 19 hours, about 19.5 hours, about 20 hours, about 20.5 hours, about 21 hours, about 21.5 hours, about 22 hours, about 22.5 hours, about 23 hours, about 23. 5 hours, about 24 hours, or about 35 hours, or about 48 hours. In some embodiments, about 10 hours, about 10.5 hours, about 11 hours,
  • the cells are incubated with the magnetic particle for about 12 hours. In some embodiments, at its widest point, the diameter of the magnetic particle is in the range of from about 20 nm to about 1 ⁇ .
  • the cell membranes are disrupted chemically with a lysis buffer comprising a detergent.
  • the detergent is a nonionic detergent.
  • the lysis buffer comprises a chaotropic salt.
  • the chaotropic salt is selected from the group of guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, lithium chloride, sodium perchlorate, sodium trichloroacetate or a mixture thereof.
  • the lysis buffer comprises guanidine isothiocyanate.
  • the lysis buffer comprises a non-chaotropic salt.
  • the non-chaotropic salt is selected from the group of sodium chloride, potassium chloride, ammonium chloride, calcium chloride, magnesium chloride or a mixture thereof.
  • the salt has a concentration of about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about 1.5M, about 2M, about 2.5M, about 3M, about 3.5MM, about 4M, about 4.5M, or about 5M.
  • a method of purifying a nucleic acid from a virus comprising: i) incubating a magnetic particle with an composition comprising intact virus, ii) disrupting the viral coat; and iii) separating the nucleic acid from viral debris and contaminants.
  • the nucleic acid is DNA or RNA.
  • the magnetic particle comprises Fe 3 C"4.
  • the magnetic particle is added at a concentration of 0.05 to 1.0 mg/ml.
  • the magnetic particle is uncoated.
  • the composition has a non-acidic pH. In some embodiments, the composition has a neutral or basic pH.
  • the composition has a pH of at least about 4.0, 5,0, 6,0 or 7.0. In some embodiments, the composition has a pH of about 4.0. In some embodiments, the composition has a pH of about 7.0. In some embodiments, the composition has a physiological pH.
  • a method of purifying a nucleic acid comprising: i) incubating a magnetic particle with an composition comprising the nucleic acid; and ii) separating the nucleic acid from the magnetic particle and contaminants; wherein the composition is non acidic.
  • the composition has a neutral or basic pH.
  • the composition has a pH of at least about 7.0. In some embodiments, the
  • the composition is at physiological pH.
  • the magnetic particle comprises Fe 3 0 4 .
  • the magnetic particle comprises Fe 2 0 3 .
  • the magnetic particle is added at a concentration of 0.05 to 1.0 mg/ml.
  • the magnetic particle is uncoated.
  • the nucleic acid is DNA, RNA or PNA.
  • the nucleic acid is RNA.
  • the RNA is an RNA selected from mRNA, miRNA, non-coding RNA (ncRNA), tRNA, rRNA, pre-mRNA, tmRNA, piRNA, CRISPR RNAs, snRNA, viral RNA, and dsRNA.
  • the nucleic acid is DNA.
  • a composition comprising: i) cellular material; and ii) a magnetic particle; wherein the composition is non-acidic.
  • cellular material refers to any substance derived from a cell, including an intact cell, cell membranes, secreted protein, etc.
  • the magnetic particle is uncoated.
  • the magnetic particle comprises Fe 3 0 4 .
  • the magnetic particle comprises Fe 2 0 3 .
  • the cellular material comprises intact cells.
  • the composition further comprises a biological medium.
  • the cellular material comprises disrupted cells.
  • the composition is neutral or basic.
  • the composition is at physiological pH.
  • the composition further comprises a chaotropic salt.
  • the chaotropic salt is selected from the group of guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, lithium chloride, sodium perchlorate, sodium trichloroacetate or a mixture thereof.
  • the chaotropic salt is guanidine thiocyanate.
  • a system comprising: i) an composition as disclosed herein; and ii) a means for immobilizing a magnetic particle.
  • the magnetic particle comprises Fe 3 0 4 .
  • the magnetic particle comprises Fe 2 0 3 .
  • the magnetic particle is uncoated.
  • the purified nucleic acid is in solution.
  • the means for immobilizing a magnetic particle is a magnetic field.
  • the magnetic field is provided by a magnet.
  • the means for immobilizing a magnetic particle comprises a filter.
  • the means for immobilizing a magnetic particle is a spin column.
  • kits for isolating a nucleic acid from an intact cell comprising: a magnetic particle; a cell lysis buffer, and a silica membrane.
  • the cell lysis buffer has a pH of about 7.0 or greater. In some embodiments, the cell lysis buffer is non-acidic. In some embodiments, the cell lysis buffer comprises guanidine isothiocyanate. In some embodiments, the kit further comprises a column, wherein the silica membrane is in the column. In some embodiments, the kit further comprises a washing solution. In some embodiments, the kit further comprises an eluting solution. In some embodiments, the magnetic particle comprises magnetite. In some embodiments, the magnetic particle comprises at least about 97% Fe 3 0 4 . In some embodiments, the magnetic particle consists essentially of magnetite. In some embodiments, the kit further comprises a magnet. In some embodiments, the magnet possesses a magnetic intensity of about 100 gauss.
  • the present invention provides a method for isolating biopolymers, particularly nucleic acids, such as DNA or RNA or hybrid molecules of DNA and RNA, from contaminants, such as other biomolecules, including proteins, monosaccharides, polysaccharides, lipids and cellular components, such as cell membranes in a medium, using magnetic particles.
  • nucleic acid solution comprises the magnetic particle and the nucleic acid.
  • the nucleic acid is DNA, RNA or PNA. In some embodiments, the nucleic acid comprises chromatin. In some embodiments, the nucleic acid comprises RNA. In some embodiments, the RNA is an RNA selected from mRNA, miRNA, non-coding RNA (ncRNA), tRNA, rRNA, pre-mRNA, tmRNA, piRNA, CRISPR RNAs, snRNA, viral RNA, and dsRNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the DNA is single stranded. In some embodiments, the DNA is double stranded. In some embodiments, the nucleic acid is selected from a plasmid and a vector.
  • the nucleic acid has been synthesized in vitro (e.g. cDNA, PCR product).
  • the nucleic acid is a mitochondrial nucleic acid.
  • the nucleic acid is a chromosomal nucleic acid.
  • the nucleic acid is a chloroplast nucleic acid.
  • the nucleic acids isolated by the methods disclosed herein are suitable, without further isolation or purification, for analysis or further processing in a downstream method that requires a nucleic acid. In some embodiments, the downstream method is selected from
  • sequencing is selected from chain termination sequencing (e.g. Sanger sequencing), next generation sequencing, high-throughput sequencing, shotgun sequencing, real-time sequencing, ion semiconductor sequencing, pyrosequencing, nanoball sequencing, and sequencing by ligation.
  • chain termination sequencing e.g. Sanger sequencing
  • next generation sequencing high-throughput sequencing
  • shotgun sequencing real-time sequencing
  • ion semiconductor sequencing pyrosequencing
  • nanoball sequencing and sequencing by ligation.
  • the nucleic acid is separated from the magnetic particle before its use in a downstream method.
  • the nucleic acid is not separated from the magnetic particle before its use in a downstream method.
  • the isolated nucleic acids are suitable for use in medical diagnosis, forensic testing, testing products for contamination, identifying pathogens, genetic testing, genetic therapy and biological research.
  • the intact cell is selected from a prokaryotic cell and a eukaryotic cell.
  • the plurality of intact cells comprises both prokaryotic cells and eukaryotic cells.
  • the eukaryotic cell is of an origin selected from a fungus, an animal or a plant.
  • the animal is a mammal.
  • the mammal is a human.
  • the intact cell is derived from a tissue.
  • the tissue is selected from vascular tissue, osteochondral tissue, epidermal tissue, muscular tissue, intestinal tissue, neuronal tissue, reproductive tissue, pancreatic tissue, ocular tissue, cartilage, skin, adipose tissue, skeletal muscle and smooth muscle.
  • the intact cell is derived from an organ.
  • the organ is selected from an ear, an eye, a nose, a brain, a thymus, a heart, a lung, a breast, a stomach, an intestine, a colon, a rectum, a pancreas, a spleen, a kidney, a liver, an ovary and a uterus.
  • the intact cell is derived from an anatomical structure.
  • the anatomical structure is selected from a sinus, a tooth, a bone, an esophagus, a trachea, a blood vessel, a diaphragm, a lymph node and a bladder.
  • the intact cell is derived from a gland.
  • the gland is selected from a thyroid gland, a prostate gland, an adrenal gland and a pituitary gland.
  • the intact cell or plurality of intact cells are selected from keratinocytes, exocrine secretory epithelial cells, hormone secreting cells, epithelial cells, neural cells, sensory cells, photoreceptor cells, muscle cells, extracellular matrix cells, blood cells, cardiovascular cells, vascular smooth muscle cells, kidney cells, pancreatic cells, immune cells, stem cells, germ cells, nurse cells, interstitial cells, stellate cells, liver cells, gastrointestinal cells, lung cells, tracheal cells, vascular cells, skeletal muscle cells, cardiac cells, skin cells, smooth muscle cells, connective tissue cells, corneal cells, genitourinary cells, mammary cells, reproductive cells, endothelial cells, fibroblasts, Schwann cells, adipose cells, bone cells, bone marrow cells, cartilage cells, pericytes, mesothelial cells, cells derived from endocrine tissue, stromal cells, lymph cells, endoderm-derived cells, ectoderm-derived cells
  • the intact cell or plurality of intact cells are selected from chondrocytes, chondroblasts, connective tissue fibroblasts, tendon fibroblasts, bone marrow reticular tissue fibroblasts, nonepithelial fibroblasts, pericytes, osteoprogenitor cells, osteoblasts, or osteoclasts or a combination thereof.
  • the intact cell or plurality of intact cells are selected from fibrocartilaginous cells.
  • the fibrocartilaginous cells are vascular.
  • the fibrocartilaginous cells are avascular.
  • the plurality of cells comprises meniscal cells.
  • the intact cell or plurality of intact cells are selected from stem cells, progenitor cells, totipotent cells, pluripotent cells, induced pluripotent stem cells, undifferentiated cells, differentiated cells, differentiating cells, trans- differentiating cells, cells from an adult, cells from a child, germ cells, circulating cells, resident cells, adherent cells, malignant cells, tumor cells, proliferating cells, quiescent cells, senescent cells, apoptotic cells, cytokine-producing cells, migrating cells and a combination thereof.
  • the plurality of intact cells comprises a combination of the cells disclosed herein.
  • the magnetic component comprises iron, nickel or cobalt. In some embodiments, the magnetic component comprises alnico, an aluminum-nickel-cobalt alloy. In some embodiments, the magnetic component comprises an alloy of a rare earth metal. In some embodiments, the rare earth metal is selected from scandium, yttrium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, homium, erbium, thulium, ytterbium, lutetium or a combination thereof. In some embodiments, the magnetic component comprises a rare earth mineral.
  • the rare earth mineral comprises lodestone.
  • the magnetic component comprises iron.
  • the magnetic component comprises a ferromagnetic material.
  • the ferromagnetic material is a soft ferromagnetic material.
  • the soft ferromagnetic material comprises annealed iron.
  • the soft ferromagnetic material comprises zinc, nickel, manganese or a combination thereof.
  • the soft ferromagnetic material is selected from manganese-zinc ferrite (Mn a Zn ( i_ a) Fe 2 0 4 ) and nickel- zinc ferrite (Ni a Zn ( i_ a) Fe 2 0 4 ).
  • the ferromagnetic material is a hard ferromagnetic material.
  • the hard ferromagnetic material comprises alnico or ferrite.
  • the hard ferromagnetic material is selected from strontium ferrite, SrFei 2 0i (SrO-6Fe 2 0 3 ), barium ferrite, BaFe ⁇ Oig (BaO-6Fe 2 0 3 ) and cobalt ferrite, CoFe 2 0 4 (CoO-Fe 2 03).
  • the magnetic component comprises a ferrite.
  • the ferrites is selected from hematite (Fe 2 03).
  • the magnetic component comprises a ferrite.
  • the ferrites is selected from magnetite (Fe 3 0 4 ). In some embodiments, the magnetic particle comprises Fe 3 0 4 . In some embodiments, the magnetic particle comprises Fe 2 03. In some embodiments, the magnetic particle consists essentially of Fe30 4 . In some embodiments, the magnetic particle consists essentially of Fe 2 03. In some embodiments, the magnetic particle is a shape selected from, but not limited to a sphere, a cube, an oval, a rod, a capsule shape, a tablet shape, a nondescript random shape. In some embodiments, the plurality of magnetic particles comprises magnetic particles of uniform shape. In some embodiments,
  • the plurality of magnetic particles comprises magnetic particles of non-uniform shapes. In some embodiments, the shape maximizes the surface areas of the magnetic particles. In some embodiments, the magnetic particle possesses a size such that their separation from the nucleic acid solution, for example by filtration or magnetic separation is not difficult. In some embodiments, the magnetic particle is not so large that its surface area is minimized or that it's not suitable for microscale operations. In some embodiments, the magnetic particle possesses a mean diameter of about 1 nm mean diameter to about 100 ⁇ mean. In some embodiments, the magnetic particle possesses a mean diameter of about 20 nm to about 1 ⁇ . In some embodiments, the magnetic particle possesses a mean diameter of about 10 nm to about 50 nm. In some
  • the magnetic particle possesses a mean diameter of about 100 nm to about 750 nm. In some embodiments, the magnetic particle possesses a mean diameter of about 500 nm. In some embodiments, the magnetic particle possesses a mean diameter of about 1 ⁇ to about 15 ⁇ . In some embodiments, the magnetic particle possesses a mean diameter of about 2 ⁇ to about 10 ⁇ . In some embodiments, the magnetic particle possesses a mean diameter of about 1 ⁇ to about 5 ⁇ . In some embodiments, the magnetic particle possesses a mean diameter of about 1 ⁇ . In some embodiments, the magnetic particle possesses a mean diameter of about 0.5 ⁇ to about 20 ⁇ at its widest point. In some embodiments, the magnetic particle comprises an uncoated magnetic particle.
  • the uncoated magnetic particle is not wholly or partially covered by an alternate material.
  • the alternate material is selected from a synthetic polymer and a biopolymer.
  • the magnetic particle is coated.
  • the magnetic particle does not comprise silicon.
  • the magnetic particle does comprise silicon. In some embodiments, the magnetic particle assists in isolating a nucleic acid in a non-acidic environment. In some embodiments, the magnetic particle assists in isolating a nucleic acid in a nearly neutral environment. In some embodiments, the magnetic particle assists in isolating a nucleic acid in a neutral environment. In some embodiments, the magnetic particle assists in isolating a nucleic acid in a basic environment. In some embodiments, the magnetic particle assists in isolating a nucleic acid in a pH of about 7.0. In some embodiments, the magnetic particle assists in isolating a nucleic acid in a pH of about 4.0.
  • the magnetic particle assists in isolating a nucleic acid in an environment having a physiological pH. In some embodiments, the magnetic particle assists in isolating a nucleic acid in a pH of 7.0, or above. In some embodiments, the magnetic particle assists in isolating a nucleic acid in a pH of more than 4.0, more than 5.5, more than 6.0, more than 6.5, more than 7.0, more than 7.5, more than 8.0, more than 8.5, or more than 9.0. In some embodiments, the magnetic particle does not bind the nucleic acid. In some embodiments the magnetic particle does not bind a cell or a component thereof.
  • a plurality of magnetic particles is combined with a plurality of intact cells at a concentration of about 0.02 mg magnetic particles/ 10 6 cells to about 4 mg magnetic particles/ 10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 0.01 mg magnetic particles/10 6 cells to about 0.2 mg magnetic particles/10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 0.1 mg magnetic particles/ 10 6 cells to about 2 mg magnetic particles/10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 0.1 mg magnetic particles/ 10 6 cells.
  • the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 0.2 mg magnetic particles/10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 0.6 mg magnetic particles/ 10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 0.8 mg magnetic particles/ 10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 1 mg magnetic particles/ 10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 1.2 mg magnetic particles/5 x 10 6 cells.
  • the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 1.4 mg magnetic particles/10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 1.6 mg magnetic particles/ 10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 1.8 mg magnetic particles/ 10 6 cells. In some embodiments, the plurality of magnetic particles is combined with the plurality of intact cells at a concentration of about 2.0 mg magnetic particles/ 10 6 cells.
  • the methods comprise combining the plurality of magnetic particles and the plurality of intact cells in an aqueous solution.
  • the aqueous solution is a biological medium.
  • the biological medium comprises a cell culture medium.
  • the methods comprise combining the plurality of magnetic particles and the plurality of intact cells in a biological medium, wherein the plurality of intact cells are adherent to a cell culture container.
  • the biological medium comprises a cell culture medium selected from Balanced Salts, Dulbecco's Modified Eagle's Medium,
  • the biological medium comprises a biological serum.
  • the biological serum is fetal bovine serum, fetal calf serum, fetal goat serum, horse serum or a combination thereof.
  • the biological serum content of the biological medium is about 0.5% v/v, about 1% v/v, about 2% v/v, about 5% v/v, about 10% v/v, about 15% v/v, about 20% v/v, about 50% v/v, about 99%) v/v, about 100%) v/v.
  • the biological medium comprises a buffering agent.
  • the buffering agent is selected from MES, ADA, PIPES, ACES, MOPSO, MOPS, BES, TES, HEPES, DIPSO, Acetamidoglycine, TAPSO, POPSO, HEPPSO, HEPPS, Tricine, Glycinamide, Bicine, TAPS or a combination thereof.
  • the biological medium comprises a growth factor, a protein, a chemical, a
  • the biological medium comprises an antibiotic and/or an antimycotic.
  • the antibiotic is penicillin, streptomycin, actinomycin D, ampicillin, blasticidin, carbenicillin, cefotaxime, fosmidomycin, gentamicin, kanamycin, neomycin, polymyxin B, or any combination thereof.
  • the antimycotic is amphotericin B, nystatin, natamycin or any combination thereof.
  • the drug is selected from an anti-inflammatory drug.
  • the anti-inflammatory drug is a non-steroidal anti-inflammatory drug.
  • the non-steroidal anti-inflammatory drug is a cyclooxygenase (COX) inhibitor.
  • COX inhibitor is selected from a COX1 inhibitor, COX2 inhibitor or combination thereof.
  • the antiinflammatory drug is a steroid. In some embodiments, the steroid is a glucocorticoid.
  • the biological medium comprises a protein, wherein the protein is selected from a growth factor, a protease, a kinase, a cytokine, a chemokine, a structural protein, a hormone, a receptor, a ligand, a channel, a nucleus or a combination thereof.
  • biological medium comprises a growth factor.
  • the growth factor is selected from Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs), Brain-derived neurotrophic factor (BDNF), Colony-stimulating factor (CSF), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GDNF), Granulocyte colony-stimulating factor (G-CSF),
  • AM Adrenomedullin
  • Ang Angiopoietin
  • BMPs Bone morphogenetic proteins
  • BMPs Brain-derived neurotrophic factor
  • CSF Colony-stimulating factor
  • EGF Epidermal growth factor
  • EPO Erythropoietin
  • FGF Fibroblast growth factor
  • G-CSF Granulocyte colony-stimulating factor
  • Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), insulin, Insulin-like growth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Thrombopoietin (TPO), Transforming growth factor alpha(TGF-a), Transforming growth factor beta(TGF-P), Tumor necrosis factor-alpha(TNF-a), Vascular endothelial growth factor (VEGF), placental growth factor (P1GF), Foetal Bovine Somatotrophin (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 or a combination thereof.
  • HGF Hepatocyte growth factor
  • HDGF Hepatoma-derived growth factor
  • IGF Insulin-like growth factor
  • biological medium comprises cellular differentiation agents, in some embodiments, cells are cultured with a cell culture supernatant or cell culture conditioned media.
  • the biological medium comprises a bodily fluid.
  • the bodily fluid is selected from blood, urine, spinal fluid, amniotic fluid, saliva, semen, pericardial fluid, peritoneal fluid, pleural fluid, synovial fluid, and a combination thereof.
  • the methods further comprise incubating the intact cell-magnetic particle composition in the biological medium for about 0.5 hours, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 1 1 hours, about 1 1.5 hours, about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours, about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours, about 16 hours, about 16.5 hours, about 17 hours, about 17.5 hours, about 18 hours, about 18.5 hours, about 19 hours, about 19.5 hours, about 20 hours, about 20.5 hours, about 21 hours, about 21.5 hours, about 22 hours, about 22.5 hours, about 23 hours, about 23.
  • the methods comprise incubating the intact cell and the magnetic particle in the biological medium over night. In some embodiments, the methods comprise incubating the intact cell-magnetic particle composition in the biological medium for about 8 hours. In some embodiments, the methods comprise incubating the intact cell-magnetic particle composition in the biological medium for about 10 hours. In some embodiments, the methods comprise incubating the intact cell-magnetic particle composition in the biological medium for about 12 hours. In some embodiments, the methods comprise incubating the intact cell-magnetic particle composition in the biological medium for about 14 hours. In some embodiments, the methods comprise incubating the intact cell-magnetic particle composition in the biological medium for about 16 hours.
  • the methods comprise incubating the intact cell-magnetic particle composition in the biological medium for about 18 hours. In some embodiments, incubating the intact cell-magnetic particle composition is carried out at a temperature selected from about 4 °C, about 10 °C, about 15 °C, about 20 °C, about 25°C, about 30 °C, about 35 °C, about 40 °C, about 45 °C, about 50°C, about 55 °C, about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C, about 85 °C, about 90 °C, about 95 °C and about 100 °C. In some embodiments, the temperature is at least about 4 °C.
  • the temperature is at least about 20 °C. In some embodiments, the temperature is about room temperature (e.g., about 25 °C). In some embodiments the temperature is about 37 °C. In some embodiments, the temperature is less than about 65 °C. Washing cells of biological medium before lysis
  • the methods further comprise isolating the intact cells and magnetic particles, for example from any biological medium.
  • the methods comprises centrifuging a culture comprising biological medium, the intact cells and the magnetic particles and washing the resulting pellet.
  • the methods comprise centrifuging the intact cell-magnetic particle composition at about 100 RPM, about 200 RPM, about 300 RPM, about 400 RPM, about 500 RPM, about 600 RPM, about 700 RPM, about 800 RPM, about 900 RPM, about 1000 RPM, about 1100 RPM, about 1200 RPM, about 1300 RPM, about 1400 RPM, about 1500 RPM, about 1600 RPM, about 1700 RPM, about 1800 RPM, about 1900 RPM, about 2000 RPM, about 2100 RPM, about 2200 RPM, about 2300 RPM, about 2400 RPM, about 2500 RPM, about 2600 RPM, about 2700 RPM, about 2800 RPM, about 2900 RPM, about 3000 RPM, about 3500 RPM, about 4000 RPM or about 5000 RPM.
  • the methods comprise centrifuging the intact cell and magnetic particle at about 1500 RPM.
  • washing the intact cell-magnetic particle composition comprises washing the intact cell-magnetic particle composition with a solution that mimics an osmolality and ion concentration of a fluid in a human body.
  • washing the intact cell-magnetic particle composition comprises contacting the intact cell-magnetic particle composition with a buffered solution.
  • washing the intact cell-magnetic particle composition comprises contacting the intact cell-magnetic particle composition with a buffered saline solution.
  • the buffered saline solution comprises phosphate buffered saline.
  • the methods comprise washing the intact cell-magnetic particle composition once. In some embodiments, the methods comprise washing the intact cell-magnetic particle composition more than one time. In some embodiments, the methods comprise washing the intact cell-magnetic particle composition about two times, about three times, about four times or about five times.
  • disrupting the cellular membrane of the intact cell to produce a magnetic particle containing cell lysate comprises a chemical disruption. In some embodiments, disrupting the cellular membrane of the intact cell to produce a magnetic particle containing cell lysate comprises contacting the intact cell-magnetic particle composition with a cell lysis buffer.
  • the plurality of intact cells possesses a concentration of about 1 cell/mL, about 10 cells/mL, about 100 cells/mL, about 1000 cells/mL, about 10,000 cells/mL, about 2 x 10 5 cells/mL, about 3 x 10 5 cells/mL, about 4 x 10 5 cells/mL, about 5 x 10 5 cells/mL, about 6 x 10 5 cells/mL, about 7 x 10 5 cells/mL, about 8 x 10 5 cells/mL, about 9 x 10 5 cells/mL, about 10 6 cells/mL, about 2 x 10 6 cells/mL, about 3 x 10 6 cells/mL, about 4 x 10 6 cells/mL, about 5 x 10 6 cells/mL, about 6 x 10 6 cells/mL, about 7 x 10 6 cells/mL, about 8 x 10 6 cells/mL, about 9 x 10 6 cells/mL, about 10 7 cells/mL, about 2 x 10 7 cells/mL, about 2 x
  • the cell lysis buffer comprises a buffered solution.
  • the cell lysis buffer comprises a lysis buffer.
  • the buffered solution comprises a buffer selected from TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, SSC, MES, Succinic acid and a combination thereof.
  • the cell lysis buffer possesses a non-acidic pH.
  • the cell lysis buffer possesses a neutral pH.
  • the cell lysis buffer possesses an acidic pH.
  • the cell lysis buffer possesses a physiological pH.
  • the cell lysis buffer possesses a pH at which a nucleic acid is stable. In some embodiments, the cell lysis buffer possesses a pH of about 4.0, about 5.0, about 6.0, about 7.0, about 8.0, about 9.0 or about 10.0. In some embodiments, the cell lysis buffer possesses a pH of about 7.0. In some embodiments, the cell lysis buffer possesses a pH greater than about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, or about 6.9.
  • the cell lysis buffer possesses a pH greater than about 7.0. In some embodiments, the cell lysis buffer possesses a pH greater than about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9 or about 8.0. In some embodiments, the cell lysis buffer possesses a pH greater than 8.0. In some embodiments, the cell lysis buffer comprises a detergent. In some embodiments, the detergent comprises sodium dodecyl sulfate. In some embodiments, the detergent comprises a dish soap. In some embodiments, the detergent comprises a non-ionic detergent.
  • the non-ionic detergent comprises a commercially available detergent sold under the trade names Tween®, Nonidet P-40, Triton X-100 or Brij®.
  • the concentration of the nonionic detergent is between about 0.05 to about 2.0 vol %. In some embodiments, the concentration of the nonionic detergent is between about 0.1% and about 1.0 vol %. In some embodiments, the concentration of the nonionic detergent is greater than about 2.0 vol %>.
  • the cell lysis buffer comprises an agent which disrupts the structure of or denatures a macromolecule such as a protein, lipid or membrane. In some embodiments, the cell lysis buffer comprises a chaotropic agent.
  • the chaotropic agent is selected from butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea, urea and a mixture thereof.
  • the cell lysis buffer comprises a salt.
  • the salt is selected from Tris-HCl and EDTA.
  • the salt is selected from a chaotropic salt.
  • the chaotropic salt is selected from guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, lithium chloride, sodium perchlorate, sodium trichloroacetate and a mixture thereof.
  • the chaotropic salt is guanidine thiocyanate.
  • the chaotropic salt is not guanidine isothiocyanate.
  • the salt is selected from a non-chaotropic salt.
  • non- chaotropic salt is selected from sodium chloride, potassium chloride, ammonium chloride, calcium chloride, magnesium chloride and a mixture thereof.
  • the cell lysis buffer comprises a chaotropic salt and a non-chaotropic salt.
  • the concentration of the salt in the cell lysis buffer is about 0.1M, about 0.2M, about 0.3M, about 0.4M, about 0.5M, about 0.6M, about 0.7M, about 0.8M, about 0.9M, about 1M, about 1.5M, about 2M, about 2.5M, about 3M, about 3.5MM, about 4M, about 4.5M, or about 5M.
  • the concentration of the salt is less than 50 mM.
  • the cell lysis buffer comprises an additive.
  • the additive is a non-ionic substance.
  • the additive is selected from ethylene glycol, tetraethylene glycol, polyalkylene glycol, cyclodextrin, carrageenan, dextran, dextran sulfate, xanthan, cellulose, hydroxypropyl cellulose, amylose, 2- Hydroxypropyl beta-cyclodextrin, agar, glycerol, polyvinyl alcohol.
  • the molecular weight of the polyethylene glycol is about 4000, about 5000, about 6000, about 7000, about 8000, about 10,000 or about 12,000.
  • the cell lysis buffer comprises polyvinylpyrrolidone (PVP).
  • the PVP is present at a concentration from about 0.05 vol. % to about 0.1 vol. %, in the cell lysis buffer.
  • contaminants are removed by PVP and ethanol precipitation, and as such, can improve R A quality and sensitivity of downstream applications with nucleic acids (i.e., PCR).
  • the cell lysis buffer does not contain polyethylene glycol.
  • the cell lysis buffer does not contain cellulose, cyclodextrin or amylose.
  • disrupting the cellular membrane comprises physical disruption.
  • the physical disruption is selected from vortexing, shaking, flicking, vibrating, inverting and a combination thereof.
  • the methods further comprise contacting a cell homogenizing column with the magnetic particle containing cell lysate to produce a homogenized magnetic particle containing cell lysate. In some embodiments, the methods further comprise passing the magnetic particle containing cell lysate through a needle to produce the homogenized magnetic particle containing cell lysate. In some embodiments, the methods further comprise adding the magnetic particle containing cell lysate to a rotor-stator homogenizer to produce the homogenized magnetic particle containing cell lysate. In some embodiments, the methods further comprise contacting the silica membrane with the homogenized magnetic particle containing cell lysate. In some embodiments, the silica membrane is in a silica membrane column.
  • the silica membrane comprises a mesh, a filter, a resin, a gel, or a matrix. In some embodiments, about 5%, about 10%, about 15%, about 20%>, about 25%, about 30%, about 35 %, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% of the silica membrane is silica.
  • the silica membrane further comprises a polymer, a fiber, a plastic, a wax, a paper or a protein. In some embodiments, the silica membrane column is attached to another silica membrane column.
  • the plurality of silica membrane columns is organized in a plate of silica membrane columns.
  • the plate of silica membrane columns comprises about 96 silica membrane columns.
  • the plate of silica membrane columns comprises about 384 silica membrane columns.
  • the plate can be centrifuged.
  • the silica membrane column possesses a volume of about 0.1 ml, about 0.2 ml, about 0.3 ml, about 0.4 ml, about 0.5 ml, about 0.6 ml, about 0.8 ml, about 1.0 ml, about 1.2 ml, about 1.5 ml, about 1.8 ml, about 2.0 ml, about 2.5 ml, about 3.0 ml, about 3.5 ml, about 4.0 ml, about 5.0 ml, about 8 ml, about 10 ml, about 15 ml, about 20ml, about 25 ml, about 30ml, about 35 ml, about 40 ml, about 45 ml, about 50 ml, about 55 ml or about 60 ml.
  • the magnetic particle containing cell lysate incubates on the silica membrane column. In some embodiments, the magnetic particle containing cell lysate incubates on the silica membrane column for about 15 seconds, about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes or about 10 minutes. In some embodiments, the methods further comprise applying a force to the silica membrane column. In some embodiments, applying the force to the silica membrane column comprises applying pressure, suction, centrifugation, vacuum, gravity or a combination thereof.
  • centrifugation comprises centrifuging the silica membrane column at about 1000 RPM, about 2000 RPM, about 3000 RPM, about 4000 RPM, about 5000 RPM, about 6000 RPM, about 7000 RPM, about 8000 RPM, about 9000 RPM, about 10,000 RPM, about 11,000 RPM, about 12,000 RPM, about 13,000 RPM, about 14,000 RPM, about 15,000 RPM, about 16,000 RPM, about 17,000 RPM, about 18,000 RPM, about 19,000 RPM, or about 20,000 RPM. In some embodiments, centrifugation comprises centrifuging the silica membrane column at about 10,000 RPM.
  • the methods further comprise contacting the homogenized magnetic particle containing cell lysate with a binding solution, wherein the binding solution promotes binding of nucleic acids to a silica membrane. In some embodiments the methods further comprise contacting the homogenized magnetic particle containing cell lysate with the binding solution prior to adding the homogenized magnetic particle containing cell lysate to the silica membrane. In some embodiments, the binding solution is an alcohol solution. In some embodiments, the methods further comprise mixing the binding solution with the homogenized magnetic particle containing cell lysate, wherein mixing is performed by a method selected from inverting, shaking, vortexing, pipetting, rotating or nutating.
  • the binding solution comprises methanol, ethanol, propanol, or isopropanol. In some embodiments, the binding solution comprises ethanol. In some embodiments, the alcohol solution comprises at least about 50% v/v, about 55% v/v, about 60%> v/v, about 70%> v/v, about 72% v/v, about 75% v/v, about 77% v/v, about 80% v/v, about 82% v/v, about 85%, about 87% v/v, about 90% v/v, about 92% v/v, about 95% v/v, about 97% v/v, or about 100% v/v alcohol in the alcohol solution. In some embodiments, the alcohol comprises at least about 90% v/v in the binding solution.
  • the methods further comprise removing cellular debris from the homogenized magnetic particle containing cell lysate. In some embodiments, the methods further comprise removing a non-nucleic acid material (e.g., peptides, proteins, oligosaccharides, lignans, small molecule natural products and other materials typically of natural origin) from the homogenized magnetic particle containing cell lysate. In some embodiments, the methods further comprise removing a non-nucleic acid material (e.g., peptides, proteins, oligosaccharides, lignans, small molecule natural products and other materials typically of natural origin) from the
  • the methods further comprise contacting the silica membrane with the homogenized magnetic particle containing cell lysate. In some embodiments, the methods further comprise contacting the silica membrane with the homogenized magnetic particle containing cell lysate and subsequently washing the silica membrane. In some embodiments, removing the cellular debris from the homogenized magnetic particle containing cell lysate comprises applying pressure, suction, centrifugation, a filter, vacuum, gravity or a combination thereof to the silica membrane. In some embodiments, the methods further comprise centrifuging the silica membrane to remove the cellular debris.
  • the methods further comprise contacting the silica membrane with a washing solution after adding the homogenized magnetic particle containing cell lysate to the silica membrane. In some embodiments, the methods further comprise adding contacting the silica membrane with the washing solution after adding the homogenized magnetic particle containing cell lysate supernatant to the silica membrane. In some embodiments, the methods further comprise contacting the silica membrane with the washing solution multiple times. In some embodiments, the methods further comprise contacting the silica membrane with the washing solution only once. In some
  • the methods further comprise contacting the silica membrane with the washing solution twice. In some embodiments, the methods further comprise contacting the silica membrane with the washing solution three times. In some embodiments, the methods further comprise contacting the silica membrane with the washing solution more than three times. In some embodiments, the methods further comprise applying pressure, suction, centrifugation, vacuum, gravity or a combination thereof to the silica membrane after adding the washing solution.
  • the methods comprise centrifuging the silica membrane at about 100 RPM, about 200 RPM, about 300 RPM, about 400 RPM, about 500 RPM, about 600 RPM, about 700 RPM, about 800 RPM, about 900 RPM, about 1000 RPM, about 1100 RPM, about 1200 RPM, about 1300 RPM, about 1400 RPM, about 1500 RPM, about 1600 RPM, about 1700 RPM, about 1800 RPM, about 1900 RPM, about 2000 RPM, about 2100 RPM, about 2200 RPM, about 2300 RPM, about 2400 RPM, about 2500 RPM, about 2600 RPM, about 2700 RPM, about 2800 RPM, about 2900 RPM, about 3000 RPM, about 3500 RPM, about 4000 RPM or about 5000 RPM to remove the cellular debris. In some embodiments, the methods comprise centrifuging the silica membrane at about 1500 RPM to remove the cellular debris.
  • the magnetic particle and the nucleic acid are removed from the cell lysis buffer comprising contaminants (e.g. cell debris, viral debris, or non-desired biomolecules) and are subsequently rinsed with the washing solution.
  • the washing solution removes cellular debris or cellular components from the nucleic acid.
  • the washing solution removes a protein from the nucleic acid.
  • the washing solution removes a lipid from the nucleic acid.
  • the washing solution removes a salt from the nucleic acid.
  • the washing solution is capable of removing contaminants (e.g., molecules other than nucleic acids) from the magnetic particles.
  • the washing solution comprises a pH buffer.
  • the pH buffer is selected from TAPS, Bicine, Tris, Tricine, TAPSO, HEPES, TES, MOPS, PIPES, Cacodylate, SSC, MES, succinic acid and a combination thereof.
  • the washing solution comprises an alcohol solution.
  • the alcohol solution comprises ethanol.
  • the alcohol solution comprises 70% ethanol/30% water (v/v), 75%) ethanol/25%) water (v/v), or 80%> ethanol/20%> water (v/v).
  • the methods comprise isolating the nucleic acids to a purity of at least 80%>, at least 90%>, at least 95%, or at least 99% or more. In some embodiments, the present methods are suitable to remove at least 80%, at least 90%, at least 95%, or at least 99% or more of a plurality of non-nucleic acid materials in the magnetic particle containing cell lysate.
  • the methods comprise eluting the plurality of nucleic acids from the silica membrane. In some embodiments, the methods comprise contacting the silica membrane with an eluting solution. In some embodiments, the eluting solution elutes the nucleic acid from the silica membrane. In some embodiments, the eluting solution does not remove the nucleic acid from the magnetic particle. In some embodiments, the eluting solution possesses a low ionic strength. In some embodiments, the eluting solution comprises a buffer. In some embodiments, the eluting solution comprises a Tris buffer.
  • the concentration of Tris is about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15mM, about 20 mM, about 25 mM, or about 50 mM. In some embodiments, the concentration of Tris is about lOmM.
  • the eluting solution comprises EDTA. In some embodiments, the eluting solution is EDTA-free. In some embodiments, the eluting solution is non-acidic. In some embodiments, the eluting solution is neutral.
  • the eluting solution possesses a pH of between about 4 and about 10.
  • the elution buffer comprises water.
  • the water is deionized water.
  • the water is nuclease free water.
  • the water is R Ase free water.
  • the water is DNAse free water.
  • the eluting solution consists essentially of water.
  • the water possesses a pH of about 4, about 4.5, about 5, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5 or about 10.
  • the water possesses a neutral pH.
  • the water possesses a pH of about 7.0. In some embodiments, the water possesses a pH of about 7.1. In some embodiments, the water possesses a pH greater than 7.0. In some embodiments, the methods further comprise incubating the eluting solution on the silica membrane. In some embodiments, the methods comprise incubating the eluting solution on the silica membrane for about 30 seconds, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes or about 10 minutes. In some embodiments, the methods comprise incubating the eluting solution on the silica membrane for a time less than a time it takes for the silica membrane to dry.
  • the methods further comprise eluting a first portion of the plurality of nucleic acids. In some embodiments, the methods further comprise applying the first portion of the plurality of nucleic acids to the silica membrane column. In some embodiments, applying the first portion of the plurality of nucleic acids to the silica membrane column serves to elute a second portion of the plurality of nucleic acids. In some embodiments, eluting the second portion of the plurality of nucleic acids with the first portion of the plurality of nucleic acids increases the concentration of the plurality of nucleic acids in the nucleic acid solution.
  • eluting the second portion of the plurality of nucleic acids with the first portion of the plurality of nucleic acids increases a yield of the plurality of nucleic acids.
  • the methods further comprise applying pressure, suction, centrifugation, vacuum, gravity or a combination thereof to the silica membrane after contacting the silica membrane with the eluting solution.
  • the methods further comprise removing the magnetic particle from the nucleic acid solution. In some embodiments, the methods further comprise removing the plurality of magnetic particles from the nucleic acid solution. In some embodiments, the methods further comprise applying a magnet to the nucleic acid solution. In some embodiments, the methods further comprise placing the nucleic acid solution in a container and applying the magnet to the container. In some embodiments, the methods further comprise applying the magnet to the container to localize the magnetic particle in an area of the container. In some embodiments, the methods further comprise immobilizing the magnetic particle with a magnetic field. In some embodiments, the magnetic field is provided by a magnet.
  • the magnet comprises a substance selected magnetitie, lodestone, cobalt, nickel, manganese, aluminum, gadolinium, dysprosium, a ceramic (e.g. ferrite) and alnico.
  • the magnet comprises an ore.
  • the magnet comprises iron ore.
  • the magnet comprises a rare-earth magnet.
  • the rare earth magnet is selected from a samarium-cobalt magnet and a neodymium-iron-boron magnet (NIB).
  • the magnet possesses a magnetic intensity of about 10 gauss, about 20 gauss, about 30 gauss, about 40 gauss, about 50 gauss, about 60 gauss, about 70 gauss, about 80 gauss, about 90 gauss, about 100 gauss, about 110 gauss, about 120 gauss, about 130 gauss, about 140 gauss, about 150 gauss, about 160 gauss, about 170 gauss, about 180 gauss, about 190 gauss, about 200 gauss, about 250 gauss, about 300 gauss, about 350 gauss, about 400 gauss, about 450 gauss, or about 500 gauss.
  • the magnet possesses a magnetic intensity of about 100 gauss.
  • immobilizing the magnetic particle comprises applying a force to the container.
  • the force is selected from gravity, centrifugation, a magnetic field or a combination thereof.
  • the methods further comprise removing a portion of the nucleic acid solution from the container.
  • the portion of the nucleic acid solution is not in contact with the magnetic particle.
  • removing the magnetic particle from the nucleic acid solution comprises filtration of the nucleic acid solution, wherein the magnetic particles are captured by a filter and nucleic acid solution passes through the filter.
  • removing the magnetic particle from the nucleic acid does not comprise applying heat, changing pH of the cell lysis buffer or changing ionic strength of the cell lysis buffer.
  • compositions comprising: i) cellular material; and ii) a magnetic particle; wherein the composition is non-acidic.
  • cellular material refers to any substance derived from a cell, including an intact cell, cell membranes, secreted protein, etc.
  • the magnetic particle is uncoated.
  • the magnetic particle comprises Fe 3 0 4 .
  • the magnetic particle comprises Fe 2 0 3 .
  • the cellular material comprises intact cells.
  • the composition further comprises cell culture medium.
  • the cellular material comprises disrupted cells.
  • the composition is neutral or basic. In some embodiments, the composition is at physiological pH.
  • the composition further comprises a chaotropic salt.
  • the chaotropic salt is selected from the group of guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, lithium chloride, sodium perchlorate, sodium trichloroacetate or a mixture thereof.
  • the chaotropic salt is guanidine thiocyanate.
  • system comprising: i) a composition as disclosed herein; and ii) a means for immobilizing a magnetic particle.
  • the magnetic particle comprises Fe 3 0 4 .
  • the magnetic particle comprises Fe 2 0 3 .
  • the magnetic particle is uncoated.
  • the purified nucleic acid is in solution.
  • the means for immobilizing a magnetic particle is a magnetic field.
  • the magnetic field is provided by a magnet.
  • the means for immobilizing a magnetic particle comprises a filter.
  • the means for immobilizing a magnetic particle is a spin column.
  • kits for isolating a nucleic acid from an intact cell comprising: a magnetic particle; a cell lysis buffer, and a silica membrane.
  • the cell lysis buffer has a pH of about 7.0 or greater.
  • the cell lysis buffer is non-acidic.
  • the cell lysis buffer comprises guanidine isothiocyanate.
  • the kit further comprises a column, wherein the silica membrane is in the column.
  • the kit further comprises a washing solution.
  • the kit further comprises an eluting solution.
  • the magnetic particle comprises magnetite.
  • the magnetic particle comprises at least about 97% Fe 3 0 4 . In some embodiments, the magnetic particle consists essentially of magnetite. In some embodiments, the kit further comprises a magnet. In some embodiments, the magnet possesses a magnetic intensity of about 100 gauss. In some embodiments, the kit further comprises a commercially available nucleic acid isolation kit or a portion or modification thereof.
  • the kit is selected from QIAamp Viral RNA Mini kit, RNeasy Mini Kit, RNeasy Micro Kit, AllPrep DNA/RNA Mini Kit, miRNeasy Mini Kit, miRNeasy Mini Kit, RNeasy Plus Mini Kit, QiAamp MinElute Virus Spin Kit, RNeasy Plus Universal Kit, QIAamp Circulating Nucleic Acid Kit, RNeasy MinElute Cleanup Kit, RNeasy Plant Mini Kit, miRNeasy Serum/Plasma Kit, RNeasy 96 Kit, QIAamp One-For-All Nucleic Acid Kit, Oligotex mRNA Mini Kit, TurboCapture 96 mRNA Kit, AllPrep DNA/RNA 96 Kit, AllPrep DNA/RNA FFPE Kit, AllPrep DNA/RNA Micro Kit, AllPrep DNA/RNA/miRNA Universal Kit, AllPrep
  • a magnet is placed below the collection tube to hold the particles at the base of the tube and the upper lysed solution is removed for the next phase of the extraction procedure.
  • an option to remove any remaining magnetite can be done by placing a magnet with at least 100 gauss at the base or side of the tube for 2 minutes. While the magnet is in place, the supernatant is removed to another tube while the magnetite remains.
  • RNA quantity and quality is measured (e.g. Nanodrop, Agilent 2100 bioanalyzer) before subsequent use in methods such as RT-PCR, microarray analysis, etc.
  • Magnetite Fe 3 0 4
  • a cell medium (1 mg/ml in 20 ml medium) and the resulting mixture is further added to cells adherent to a culture flask.
  • Cells are incubated with Fe 3 0 4 over night or for at least 12 hours.
  • Cells are harvested using accutase or trypsin and washed with PBS.
  • RNA is isolated with a Qiagen RNeasy® kit as described in Example 1 from this point without adding more Fe 3 0 4 .
  • RNA yields were higher in pellets or alginate constructs (cells that are suspended in alginate solution and crosslinked to form a gel) containing the particles compared to the controls.
  • Adherent cells human chondrocytes 0.5xl0 6 cells per extraction
  • Adherent cells human chondrocytes 0.5xl0 6 cells per extraction
  • flasks were harvested after treating with accutase, washed by PBS and centrifuged in a tube to pellet.
  • the remaining cells and particles were then lysed in 350ul of RLT lysis buffer with beta mercaptoethanol as directed by the Qiagen protocol. This lysed mix of cells and particles in the lysis buffer was then pipetted into a Qiashredder column and the RNA extraction protocol outlined by the RNeasy kit was followed. At the elution step, 100 ⁇ of RNAse free water was added to the column. From the 100 ⁇ , 50 ⁇ was removed and placed into another tube. One set of 50 ⁇ was placed on top of a magnet for 3 minutes and 25 ⁇ of this was removed from the upper section. The (-) samples in Table 1 correspond to the 25 ⁇ that was removed from the top of the 50 ⁇ after placing on a magnet (#0082). The (+) samples in Table 1 correspond to the 25 ⁇ left behind. This demonstrated that the presence of magnetite did not interefere with the reading of R A concentration. All samples were then measured using a Nanodrop.
  • Table 1 shows the concentration of RNA obtained with different concentrations of MagN97 in comparison to controls. For 0.5xl0 6 cells (in each condition), at least a 2-fold increase in RNA yield was observed with similar quality (260/280 ratio).

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Abstract

L'invention concerne des procédés permettant d'isoler des acides nucléiques comprenant l'utilisation d'une particule magnétique.
PCT/US2014/024900 2013-03-14 2014-03-12 Procédés permettant d'isoler des acides nucléiques WO2014159719A1 (fr)

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JP7565744B2 (ja) 2014-10-24 2024-10-11 アヴェクタス リミテッド 細胞原形質膜を越える送達方法
JP2022180643A (ja) * 2014-10-24 2022-12-06 アヴェクタス リミテッド 細胞原形質膜を越える送達方法
JP2021007404A (ja) * 2014-10-24 2021-01-28 アヴェクタス リミテッド 細胞原形質膜を越える送達方法
US10337001B2 (en) 2014-12-03 2019-07-02 Agilent Technologies, Inc. Guide RNA with chemical modifications
US10900034B2 (en) 2014-12-03 2021-01-26 Agilent Technologies, Inc. Guide RNA with chemical modifications
US11306309B2 (en) 2015-04-06 2022-04-19 The Board Of Trustees Of The Leland Stanford Junior University Chemically modified guide RNAs for CRISPR/CAS-mediated gene regulation
US11851652B2 (en) 2015-04-06 2023-12-26 The Board Of Trustees Of The Leland Stanford Junior Compositions comprising chemically modified guide RNAs for CRISPR/Cas-mediated editing of HBB
US11535846B2 (en) 2015-04-06 2022-12-27 The Board Of Trustees Of The Leland Stanford Junior University Chemically modified guide RNAS for CRISPR/Cas-mediated gene regulation
US10767175B2 (en) 2016-06-08 2020-09-08 Agilent Technologies, Inc. High specificity genome editing using chemically modified guide RNAs
US20190153427A1 (en) * 2016-07-08 2019-05-23 President And Fellows Of Harvard College Determination of rna in blood or other fluids
CN108031442B (zh) * 2017-12-29 2020-12-18 猎源（上海）生物医药科技有限公司 一种纳米脂质磁球及其制备方法
CN108031442A (zh) * 2017-12-29 2018-05-15 猎源（上海）生物医药科技有限公司 一种纳米脂质磁球及其制备方法、分离提取游离dna的试剂盒及其应用
CN111249479B (zh) * 2020-03-02 2021-09-28 同济大学 一种仿生纳米材料及其制备方法和其在对动脉粥样硬化成像的药物中的用途
CN111249479A (zh) * 2020-03-02 2020-06-09 同济大学 一种仿生纳米材料及其制备方法和其在对动脉粥样硬化成像的药物中的用途
US11884915B2 (en) 2021-09-10 2024-01-30 Agilent Technologies, Inc. Guide RNAs with chemical modification for prime editing
CN114213492A (zh) * 2021-09-23 2022-03-22 武汉奥科鼎盛生物科技有限公司 一种dna纯化试剂、其制备方法及dna纯化方法
CN116053017A (zh) * 2022-11-04 2023-05-02 医波(厦门)科技有限公司 一种复合磁性微球及其制备方法和应用
CN116053017B (zh) * 2022-11-04 2023-08-22 医波(厦门)科技有限公司 一种复合磁性微球及其制备方法和应用

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