WO2016075701A2 - Procédé d'extraction d'adn faisant appel à des nanoparticules magnétiques nues - Google Patents

Procédé d'extraction d'adn faisant appel à des nanoparticules magnétiques nues Download PDF

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WO2016075701A2
WO2016075701A2 PCT/IN2015/000416 IN2015000416W WO2016075701A2 WO 2016075701 A2 WO2016075701 A2 WO 2016075701A2 IN 2015000416 W IN2015000416 W IN 2015000416W WO 2016075701 A2 WO2016075701 A2 WO 2016075701A2
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dna
extraction
buffer
magnetic nanoparticles
magnetic
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PCT/IN2015/000416
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WO2016075701A3 (fr
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Aniruddha BHATI
Sajesh Puthenpurackal KRISHNANKUTTY
Nanappan Ramchand CHANIYILPARAMPU
Santhosh SREEDHAR
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Scigenom Labs Pvt. Ltd.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • This invention relates to a method for extraction of DNA using naked magnetic nanoparticles. More particularly, the invention relates to a method for DNA extraction from various biological systems using naked magnetic nanoparticles with high efficiency and purity.
  • DNA extraction has been a tool used extensively across the world in applied sciences. Most of the research breakthrough in biological research in the previous years directly or indirectly banked on DNA extraction. Applied research; be it Cloning or sequencing involves the use of DNA extraction methodologies. It was in the year of 1869 when Swiss scientist Friedrich Miescher first reported the extraction of DNA during a study conducted to elucidate the building blocks of life. Since then the science of extraction has advanced and has now reached its peak. A number of methods are available for DNA extraction in the literature viz. Phenol-chloroform, Benzyl alcohol method, Gradient based strategies, alkaline lysis method and so on. The newer approach in this regard is the solid phase extraction strategies.
  • Solid-phase mentioned herein is primarily a physical moiety like glass beads, silica matrices etc. on which the DNA can bind and can be subsequently extracted. Recent advancements in this field involve the use of magnetisable solid phase support. This also serves an important purpose for the ease of extraction in the sense that the centrifugation step can be averted.
  • US Patent 5512439 discloses a method where oligonucleotides can be reversibly attached to microspheres impregnated with iron oxide and thereby target nucleic acids to be separated out from the surrounding media.
  • US Patent 6027945 describes the use of magnetic silica particles with size ranging a few microns to extract and purify nucleic acids including DNA fragments, plasmid DNA, and R A.
  • WO 96/18731 by Deggerdal el. al. discloses a method where nucleic acid is extracted in the presence of a solid support by treating the samples with a detergent. The process results in the binding of DNA present in the sample to the support which can later be eluted out.
  • Another objective of the invention is to provide a method, where the damage done to the DNA during extraction is reduced to a bare minimum and further avoids the use of instruments like centrifuge etc. Summary of the invention:
  • the present invention provides a method for the extraction of DNA from biological samples using naked magnetic nanoparticles with the help of a magnetic solid support which is preferably (but not limited to be) uncoated.
  • the magnetic nanoparticle used according to the invention can be synthetic analogues of any suitable magnetic material or combination of materials, such as magnetite, ulvospinel, hematite, ilmenite, maghemite, jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite,lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite, wairauite, or any combination thereof. It can also be made up of transition metal such as iron, manganese, nickel, cobalt, zinc, etc. These magnetic particles can be of various shapes and sizes as the extraction is not dependent on any of these parameters.
  • the method disclosed in the present invention makes use of a proprietary mix of extraction solution which comprises magnetic nanOparticles in an amount of 1 to 3 mg/ml together with binding buffer consisting of monovalent salt in a concentration of 1 to 5 moles and PEG-8000 in an amount of 5 to 25% w/w of the total mixture.
  • the use of the extraction solution separates the DNA from all proteins and hemoglobin thereby yield the resultant DNA with good yields and great purity.
  • the current method can extract DNA from a number of biological samples including but not limited to Blood, Plant tissues, Animal tissues, Bacteria, Viruses, Fungi, Saliva, Urine, Stool, Semen, CSF, Paraffin fixed tissues, Tears, Amniotic fluid, Soil etc.
  • the method makes use of specialized lysis buffer to lyse the cellular components for effectively exposing the DNA to magnetic support.
  • the method also involves components which bind the DNA reversibly to magnetic support that could be effectively removed later for downstream processing like PCR amplification, Sequencing etc.
  • the method in another aspect contains special wash solutions and conditions whereby minimal contaminants are bound to the magnetic particles. Concurrently, the contaminants, if any, are effectively washed off from the magnetic nanoparticles without the loss of bound DNA on it.
  • Figure 1 General protocol for the extraction of DNA from different biological systems using uncoated magnetic nanoparticles
  • Figure 2 DNA extracted from FFPE samples (Lane 1 , 2, 3 and 4 represent extraction from 3 different FFPE Samples)
  • Figure 3 DNA extracted from Fecal samples (Lane 1 and 2 represent extractions from fecal in duplicates)
  • Figure S DNA extracted from Bacterial samples (Lane 1 , 2, 3, and 4 represent extraction from different bacteria)
  • Figure 7 Sequencing chromatogram of the PCR product amplified from extracted DNA using Sanger sequencing technology
  • Figure 8 DNA extracted from Blood samples(Lane 1 , 2, 3 and 4 represent extraction from blood collected from 4 individuals)
  • Figure 10 DNA extracted from ⁇ 55 ⁇ samples(Lane 1 , 2, 3, and 4 represent extractions from different tissues)
  • FIG. 11 PCR amplification of Tissue samples
  • FIG. 12 DNA extracted from Saliva samples(Lane 1, 2, 3 and 4 represent extraction from saliva collected from 4 individuals)
  • Figure 14 Comparison of four different Tissue DNA samples extracted using Magnetic nanoparticle based method (to the left of the marker) and using conventional method (to the right of the marker)
  • Figure 15 Comparison of four different Plant DNA samples extracted using Magnetic nanoparticle based method (to the left of the marker) and using conventional method (to the right of the marker)
  • Figure 16 Comparison of four different Blood DNA samples extracted using Magnetic nanoparticle based method (to the left of the marker) and using conventional method (to the right of the marker)
  • Figure 17 Comparison of four different Bacterial DNA samples extracted using Magnetic nanoparticle based method (to the left of the marker) and using conventional method (to the right of the marker)
  • Figure 18 depicts comparison of DNA extracted from blood samples using (a) Method mentioned in GB 2455780 patent and DNA extracted using the (b) present method (All proteins and haemoglobin removed
  • Figure 19 depicts comparison of DNA Purity and yield of DNA extracted from Blood using (a) Method mentioned in GB 2455780 patent and (b) DNA extracted using the present method (All proteins and haemoglobin removed), which indicates that the present method gives recovery almost double than the method disclosed in GB'780.
  • the 260/280 and 260/230 ratios of around 1.8 shows pure DNA and not contaminated by RNA and/or proteins.
  • Figure 20 depicts DNA extracted using (a) Method mentioned in GB 780 (2009) patent and DNA extracted using the (b) present method (higher recovery)
  • the instant invention provides an efficient method for extracting DNA from various biological samples like Formalin fixed and paraffin embedded (FFPE), Fecal, Bacteria, Blood, Plant tissue, animal Tissues, Viruses, Fungi, Saliva, Urine, Stool, Semen, CSF, Paraffin fixed tissues, Tears, Amniotic fluid, Soil etc. using naked magnetic nanoparticles.
  • FFPE Formalin fixed and paraffin embedded
  • Fecal Bacteria
  • Blood Blood
  • Plant tissue animal Tissues
  • Viruses Fungi
  • Saliva Saliva
  • Urine Urine
  • Stool Semen
  • CSF Paraffin fixed tissues
  • Tears Amniotic fluid, Soil etc.
  • the method disclosed in the present invention makes use of a proprietary mix of extraction solution which comprises magnetic nanoparticles in an amount of 1 to 3 mg/ml together with binding buffer consisting of monovalent salt in a concentration of 1 to 5 moles and polymer such as PEG-8000 in an amount of 5 to 25% w/w of the total mixture.
  • binding buffer consisting of monovalent salt in a concentration of 1 to 5 moles and polymer such as PEG-8000 in an amount of 5 to 25% w/w of the total mixture.
  • the lysis buffer contains combinations of various detergents such as alkali metal alkylsulphate salt, Urea and Tween 20 or combinations thereof.
  • alkali metal alkylsulphate salt is sodium dodecyl sulphate.
  • One preferred lysis buffer for a biological sample of Blood comprises of Sodium dodecyl sulphate and Tween 20.
  • the incubation for lysis typically takes 3-5 minutes.
  • the lysed tissue optionally incubated with Proteinase or RNase enzymes or both.
  • the present invention encompasses the proprietary mix of extraction solution which comprising of magnetic nanoparticles and binding buffer consisting of a monovalent salt and a polymer.
  • the proprietary mix of extraction solution makes the process more efficient and contamination free.
  • the magnetic nanoparticles are preferably magnetite (Fe304) while the binding buffer contains polyethylene glycol and a monovalent salt.
  • the magnetic nanoparticles of size within 10-lOOnm are more readily available for binding the DNA and the action is more specific in the sense that it prevents non-specific adsorption of other cellular biomolecules.
  • the naked magnetic nanoparticles are selected from the group consisting of oxides of synthetic analogues of any suitable magnetic material or combination of materials, such as magnetite, ulvospinel, hematite, ilmenite, maghemite, jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite, lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite and wairauite.
  • any suitable magnetic material or combination of materials such as magnetite, ulvospinel, hematite, ilmenite, maghemite, jacobsite, trevorite, magnesioferrite, pyrrhotite, greigite, troilite, goethite, lepidocrocite, feroxyhyte, iron, nickel, cobalt, awaruite and wairauite.
  • the naked magnetic nanoparticles used, according to the invention are preferably divalent or trivalent iron oxide.
  • One preferred iron oxide nanoparticles are magnetite (Fe30,() magnetic nanoparticles, preferably of size ranging from 10-lOOnm.
  • the magnetic nanoparticles are preferably uncoated.
  • the binding buffer contains polyethylene glycol and a monovalent salt.
  • the monovalent salt used in the binding buffer for the extraction of DNA is selected from the group consisting of Sodium chloride, lithium chloride, Potassium chloride or a mixture thereof.
  • the monovalent salt is used in 1 to 5 molar concentration of the mixture.
  • the polyethylene glycol or polyoxyethylene-8000 (POE) is used in the concentration between 5 to 25 % of the mixture.
  • the proprietary mix of extraction solution comprises:
  • the pH of the extraction solution is maintained in the range of 6.0 to 8.0 ⁇ 0.2.
  • the proprietary mix of extraction solution comprises: a) Magnetic nanoparticles 1 to 3 mg/ml;
  • the pH of the extraction solution is maintained at 8.0 ⁇ 0.2.
  • the recovery of the DNA is higher in the case of the method disclosed in the invention over the method disclosed in the patent GB 2455780.
  • a comparative example of the method of GB'780 vis-a-vis the present invention that employs the specific proprietary mix of extraction solution for the recovery of pure DNA from blood sample without protein and haemoglobin contamination is described in figures 18.
  • the spectra and 260/280 and 260/230 ratios are ideal in the case of present method.
  • the comparison between the yields of the present and the method disclosed in GB'780 is shown in figure 20.
  • Effective removal of protein is shown in figure 18 and spectra with 260/280 and 260/230 ratios are shown in figure 19.
  • the specific proprietary mixture removes proteins, haemoglobin, RNA and small molecules, especially in blood samples the haemoglobin contamination is completely removed as shown in figure 20.
  • the extraction of lysed DNA using proprietary mix of extraction solution and the components with specific concentration according to the invention reduces the damage done to the DNA during extraction and thus results in good yield and purity.
  • the present invention further eliminates the use of centrifugation instruments etc.
  • An important feature of the invention includes, the provision of proprietary mix of magnetic nanoparticles and the binding buffer together in a proprietary mix which makes the process more efficient and contamination free.
  • the magnetic nanoparticles( of size within 10- 1 OOnm) are more readily available for binding the DNA and the action is more specific in the sense that it prevents non-specific adsorption of other cellular biomolecules.
  • the specialized two part wash buffer used in the instant invention removes contaminants and keeps the DNA tightly bound to the nanoparticles concurrently.
  • the wash buffers used in the invention are based on ethanol and other organic solvents such as acetone.
  • the elution buffer according to the invention may be, for example, TE buffer (Tris-EDTA buffer) or nuclease free water.
  • the method for extracting DNA from a biological sample is initiated with the addition of a suitable lysis buffer.
  • the lysis buffer depends on the type of sample used.
  • the lysis buffer contains a combination of detergents preferably Sodium dodecyl sulphate and Tween 20.
  • Post to the addition of the lysis buffer there is an incubation for some time, preferably 3 to 5 minutes.
  • the binding buffer containing the magnetic nanoparticles is added to the system thereafter.
  • the binding buffer forces the system towards a hydrophobic milieu thereby causing the DNA to precipitate.
  • the magnetic nanoparticles present in the system therefore serves as a site for this adsorption.
  • the magnetic nanoparticles and the binding buffer are clubbed together in a proprietary mix which makes the process more efficient and contamination free.
  • the magnetic nanoparticles are preferably magnetite (Fe 3 0 4 ) while the binding buffer contains polyethylene glycol and a monovalent salt. Therefore the magnetic nanoparticles (Size within 10-lOOnm) are more readily available for binding the DNA and the action is more specific in the sense that it prevents non-specific adsorption of other cellular biomolecules. This step is followed by subjecting the system to an external magnetic field where the pellet of DNA-adsorbed nanoparticles settle down under this effect and the supernatant containing the contaminants can be subsequently discarded.
  • the pellet is formed at the bottom or the side of the tube depending on the direction of the field.
  • the pellet still needs to be washed thoroughly to obtain an ultra-pure DNA.
  • the adsorbed DNA gets detached easily. This can cause a 30 to 70 % decrease in the yield.
  • a specialized wash buffer is used to remove contaminants and keeps DNA tightly bound to the nanoparticles concurrently.
  • the wash buffer used in the invention is a two part wash buffer in which one contains 80% ethanol and other contains ice-cold acetone.
  • the DNA can be subsequently eluted from the nanoparticles in an elution buffer by subjecting the pellet with hydrophilic conditions.
  • the elution buffer is TE buffer or nuclease free water.
  • the invention provides a method for extraction of DNA from Formalin fixed paraffin embedded tissues (FFPE) using magnetic nanoparticles that gives DNA with high integrity and purity which comprises:
  • Proteinase enzymes c) treating the lysed sample with proprietary mix of extraction solution according to claim 1, to precipitate DNA and thereby allowing the magnetic particles to capture on same;
  • the invention provides a method for extraction of DNA from Faecal and Soil samples using magnetic nanoparticles that gives DNA with high integrity and purity which method comprises;
  • the lysis buffer used in accordance with the invention comprises alkali metal alkylsulphate salt, more preferably sodium dodecyl sulphate.
  • the invention provides a kit for extraction of ultra-pure DNA with high yield from a biological sample which comprises;
  • Elution buffer consisting of TE buffer or nuclease free water.
  • Formalin fixation and paraffin embedding is a standard method for long-term preservation of most archived pathological specimens. Such samples provide an invaluable source for subsequent molecular studies of clinical phenotypes, especially genetic studies in which DNA is not available from fresh or frozen tissue(s) because the subjects are no longer alive. FFPE tissue is an excellent source of DNA, but its extraction remains a challenge. Formaldehyde, the effective component of formalin, leads to the generation of cross-linking between nucleic acids and proteins, and causes nucleic acids to fragment because of fixation process conditions.
  • tissue cores or microdissected tissue are subjected to xylene treatment; which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes.
  • lysis buffer which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion.Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase during the incubation period of 30minutes.
  • the tissue can be subjected to the protocol mentioned in Figure 1 which leads to a shear free high recovery of DNA.
  • the binding buffer which contains the nanoparticles, efficiently bind the entire DNA and subsequently releases it later in the elution buffer. Elution of DNA is done at 90 °C. The results for extraction from different FFPE samples are depicted in Figure 2.
  • Fecal sampling is regarded as an effective, non-invasive and easy method for various genetic and ecological research.
  • the samples are to be lysed in a suitable lysis buffer containing combination of detergents preferably Sodium dodecyl sulphate and Tween 20.
  • Protein and RNA contaminants are removed by incubating the sample with Proteinase K and RNase A respectively. Lysis is followed by incubation with iron magnetic nanoparticles and washing using suitable wash buffers, after which the bound DNA is eluted by adding TE buffer/Nuclease free water with heating at the temperature ranging from 50 °C to 60°C.
  • the result of extraction is shown in Figure 3.
  • the PCR amplification of extracted DNA samples have proved that the extracted DNA is amenable to downstream appIications.
  • the amplification results are shown in Figure 4.
  • Blood is used as an effective source for the extraction of DNA. Tn spite of being a consistent system, a number of challenges remain for efficient extraction of DNA. Various complexities like large amount of protein content, presence of RBCs etc poses major issues and demands a potential protocol for isolation. The highlight of the study remains mainly in the simplicity of the method, time, cost, number of reagents used, instrumentation etc.
  • the environment created by the buffering systems ensures the molecular integrity and intactness of the extracted DNA along with purity and concentration.
  • the buffers constitute a suitable chelating agent, surfactants and detergents which help in membrane solublization, digestion etc.
  • a starting sample volume of 200 ⁇ 1 of blood is used.
  • a buffer solution containing EDTA is used for the removal of RBCs.
  • the WBC lysis is attained by the addition of detergents like SDS, Tween-20 and Urea followed by incubation for efficient digestion.
  • a binding solution with specific mixture of PEG-8000 and 4M NaCl is used for the suspension of magnetic nanoparticles (1 to 3 mg ml) which creates a hydrophobic environment for DNA precipitation. Washing is carried out using 80% Ethanol and Acetone followed by sample elution by adding TE buffer/nuclease free water with heating at the temperature ranging from 50 °C to 60°C;
  • the method yielded a DNA concentration of about 4 ⁇ g with a 200/280 ratio ranging between 1.7 and 1.9 which is shown in Figure 8.
  • Figure 9 shows amplification using Human primers.
  • Tissue DNA isolation protocol helps in the isolation of DNA from about 50-100mg of starting material.
  • tissue homogenization can be adopted like crushing the tissue in liquid nitrogen, mincing or even directly dropping the sample in the lysis buffer. The method of homogenization determines the time taken for extraction. Lysis is facilitated by a suitable buffer containing Tris-HCl, EDTA, CaC3 ⁇ 4, SDS, and Urea. Enzymatic digestions using RNase A and Proteinase K is also adopted for better yields and purity.
  • a magnetic nanoparticle binding solution comprising of PEG-8000 and NaCl is added to the digested sample facilitating DNA binding. Washes using 80% Ethanol is carried out to remove any salts and other contaminants.
  • DNA is finally eluted by adding TE buffer/nuclease free water with heating at the temperature ranging from 50 °C to 60°C Depending on the tissue used, the yield of DNA varies from sample to sample which ranges between 20-40 ⁇ g of DNA with a purity of 1.7-1.9 which is shown in Figure 10. Amplification using Human primers is shown in Figure 11.
  • Example 6 Amplification using Human primers is shown in Figure 11.
  • DNA isolation from saliva has been regarded as a non-invasive yet efficient way of DNA extraction.
  • a large number of studies selectively prefer saliva as the source for DNA isolation.
  • the protocol followed here is relatively simple and quick and yields considerable concentration of DNA with good ⁇ .500 1
  • Saliva sample is incubated in a lysis buffer containing Ca 3 ⁇ 4, SDS and Tris-HCl.
  • the cellular contents are again incubated with magnetic nanoparticles in a buffer medium containing PEG-8000 and NaCl.
  • the DNA bound magnetic nanoparticles are washed using 80% Ethanol and Acetone, by adding TE buffer/nuclease free water with heating at the temperature ranging from 50 °C to 60°C
  • the eluted DNA was found to be around 2 g in concentration with a purity ratio between 17.-1.9 which is shown in Figure 12.
  • Amplification using Human primers is shown in Figure 13.
  • the current method serves as an excellent approach for quick and efficient extraction of DNA from various biological systems like Bacteria, Fecal, FFPE, Blood, Plants, Tissues, Viruses, Fungi, Saliva, Urine, Stool, Semen, CSF, Tears, Amniotic fluid, Soil etc.
  • the method is based mainly on effectively lysing the cells followed by addition of specific magnetic nanoparticle containing binding buffer. This provides conditions where DNA specifically binds to the naked nanoparticles effectively.
  • the DNA is finally eluted with high quantity and high purity in the elution buffer.
  • Efforts to extract DNA from Soil, Water and also purifying the desired range of DNA fragment have also found success.
  • the protocol follows the same principle of extraction where modifications are made in certain steps depending on the sample to be processed.
  • the sample is subjected to heat shock followed by enzyme treatments using Proteinase K and RNase A.
  • the rest protocol, i.e, binding and subsequent washes remain the same.
  • the DNA sample to be purified is mixed with appropriate binding buffer containing nanoparticles.
  • the concentration of magnetic nanoparticles is characteristic of the size of the DNA fragment to be purified.

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Abstract

L'invention concerne un procédé d'extraction d'ADN faisant appel à des nanoparticules magnétiques nues. L'invention concerne, plus particulièrement, un procédé d'extraction d'ADN hors de divers systèmes biologiques à l'aide de nanoparticules magnétiques nues, se caractérisant par sa grande efficacité et par la grande pureté de l'ADN extrait.
PCT/IN2015/000416 2014-11-11 2015-11-11 Procédé d'extraction d'adn faisant appel à des nanoparticules magnétiques nues WO2016075701A2 (fr)

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CN106350509A (zh) * 2016-10-18 2017-01-25 哈尔滨博泰生物科技有限公司 一种快速高效的唾液dna提取试剂盒及提取方法
CN109055359A (zh) * 2018-08-30 2018-12-21 苏州百源基因技术有限公司 一种核酸提取试剂盒及核酸提取方法
WO2016075701A3 (fr) * 2014-11-11 2019-01-03 Scigenom Labs Pvt. Ltd. Procédé d'extraction d'adn faisant appel à des nanoparticules magnétiques nues
CN109182327A (zh) * 2018-08-30 2019-01-11 尤崇革 磁性纳米粒子在核酸提取中的应用及其制备方法
CN111154750A (zh) * 2020-01-13 2020-05-15 苏州行知康众生物科技有限公司 一种针对性富集血浆目标游离dna的方法
CN112501161A (zh) * 2020-12-23 2021-03-16 华南师范大学 一种双磁粒介入的dna提取纯化方法
CN113832146A (zh) * 2021-11-15 2021-12-24 复旦大学附属中山医院 一种福尔马林固定石蜡包埋样本提取病原微生物核酸的方法
WO2022003723A1 (fr) * 2020-06-30 2022-01-06 Maggenome Technologies Pvt. Ltd. Procédé pour l'extraction d'acides nucléiques à partir d'un échantillon biologique
CN116656670A (zh) * 2023-06-29 2023-08-29 上海迈其生物科技有限公司 一种适用于植物样本的总dna的快速抽提方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5705628A (en) * 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles
US20150252407A1 (en) * 2014-03-09 2015-09-10 Nvigen, Inc. Nanostructure and methods of nucleic acid isolation
WO2016075701A2 (fr) * 2014-11-11 2016-05-19 Scigenom Labs Pvt. Ltd. Procédé d'extraction d'adn faisant appel à des nanoparticules magnétiques nues

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016075701A3 (fr) * 2014-11-11 2019-01-03 Scigenom Labs Pvt. Ltd. Procédé d'extraction d'adn faisant appel à des nanoparticules magnétiques nues
CN106350509A (zh) * 2016-10-18 2017-01-25 哈尔滨博泰生物科技有限公司 一种快速高效的唾液dna提取试剂盒及提取方法
CN109055359A (zh) * 2018-08-30 2018-12-21 苏州百源基因技术有限公司 一种核酸提取试剂盒及核酸提取方法
CN109182327A (zh) * 2018-08-30 2019-01-11 尤崇革 磁性纳米粒子在核酸提取中的应用及其制备方法
CN109055359B (zh) * 2018-08-30 2023-04-07 苏州百源基因技术有限公司 一种核酸提取试剂盒及核酸提取方法
CN109182327B (zh) * 2018-08-30 2023-04-07 苏州百源基因技术有限公司 磁性纳米粒子在核酸提取中的应用及其制备方法
CN111154750A (zh) * 2020-01-13 2020-05-15 苏州行知康众生物科技有限公司 一种针对性富集血浆目标游离dna的方法
WO2022003723A1 (fr) * 2020-06-30 2022-01-06 Maggenome Technologies Pvt. Ltd. Procédé pour l'extraction d'acides nucléiques à partir d'un échantillon biologique
CN112501161A (zh) * 2020-12-23 2021-03-16 华南师范大学 一种双磁粒介入的dna提取纯化方法
CN112501161B (zh) * 2020-12-23 2023-03-28 华南师范大学 一种双磁粒介入的dna提取纯化方法
CN113832146A (zh) * 2021-11-15 2021-12-24 复旦大学附属中山医院 一种福尔马林固定石蜡包埋样本提取病原微生物核酸的方法
CN116656670A (zh) * 2023-06-29 2023-08-29 上海迈其生物科技有限公司 一种适用于植物样本的总dna的快速抽提方法

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