WO2020159461A2 - Procédé d'isolement rapide et de haute qualité d'adn génomique ou libre circulant dans un seul tube et kit associé - Google Patents
Procédé d'isolement rapide et de haute qualité d'adn génomique ou libre circulant dans un seul tube et kit associé Download PDFInfo
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- WO2020159461A2 WO2020159461A2 PCT/TR2020/050048 TR2020050048W WO2020159461A2 WO 2020159461 A2 WO2020159461 A2 WO 2020159461A2 TR 2020050048 W TR2020050048 W TR 2020050048W WO 2020159461 A2 WO2020159461 A2 WO 2020159461A2
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- dna
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- isolation
- dna isolation
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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
Definitions
- Invention relates to a method that allows high-quality DNA isolation in a short time without the need for preliminary preparation of biological materials which exists as single cell suspension and contains cell-free DNA, and a product or kit that enables this method to be applied quickly and simply.
- the present invention relates to a method that allows DNA isolation by means of silica matrix by performing the cell lysate or homogenate preparation steps required before DNA isolation, in a single tube with optimized solutions.
- DNA isolation is known as the process of revealing the DNA molecule in the cells with specially developed techniques. DNA isolation; it is a necessary preliminary step to perform molecular tests used in diagnostic and research studies in many fields such as molecular immunology, forensic medicine, molecular genetics, molecular microbiology, veterinary medicine, and plants. To perform this process, the cell wall (membrane) must be broken down, the DNA protein complex must be dissolved and separated from molecules.
- the first operation is to break down the cell membranes and to release the cell contents containing DNA into the solution. This destruction is generally carried out by mechanically, enzymatically or chemically such as alkaline lysis, hypotonic lysis, the usage of ammonium hydrochloride and, it must be completed outside of the DNA isolation process.
- DNA isolation Another important process in the DNA isolation is the removal of protein and lipids in the environment without damaging the nucleic acids.
- the relevant process is provided with various solutions. However, already there is no any practical solution for allowing removal of erythrocytes, fragmentation of membranes, process intended to lipids and proteins, to be performed in a single tube.
- the most conventional method used for DNA isolation is the isolation of DNA with phenol-chloroform method by using enzymes such as proteinase K. Although good quality DNA is isolated as a result of this conventional process, carrying out of this method takes very long time. As a result, employees working in the laboratory are exposed to chemicals harmful to human health for a long time. This method, as well as being impractical, has harm to the health of the practitioners.
- heating method at high temperatures is used for removing the proteins from DNA. This method increases process steps and requires additional time.
- Aim of the invention is to present a method and a kit that allow high-quality DNA isolation from the samples existing as cell suspension and from the liquids containing cell-free DNA without requiring erythrocytes and cell lysis.
- Aim of the invention is to present a method that allows DNA isolation via silica matrix without performing the preliminary steps of the cell lysate or homogenate process, that are required to be applied prior to DNA isolation.
- Another aim of the invention is to obtain pure DNA without any damage and to provide time-saving by performing the removal of erythrocytes, fragmentation of membranes, process intended to lipids and proteins via reactions occurring in a single tube.
- Another aim of the invention is to provide quick, practical and pure DNA isolation that can serve more than one area with a single tube, thanks to effectiveness in DNA collection from the solutions containing cell-free DNA and in removing of proteins in said solutions through the content of single tube.
- Another aim of the invention is to present a DNA isolation method which includes the steps; protein fragmentation, DNA binding with untreated silica matrix, removing the proteins from the environment, carrying out the washing processes, performing DNA precipitation processes, DNA melting and obtaining, and a kit therefore.
- Another object of the invention is to present a versatile method that can be used easily for DNA isolation.
- the present invention presents a method and kit (product) that allow DNA isolation via silica matrix in a single tube without requiring the preliminary steps of the cell lysate or homogenate process, that are required to be applied prior to DNA isolation.
- DNA isolation method high-quality DNA is also obtained from microorganisms such as bacteria species, fungi species as well as biological materials such as whole blood, isolated leukocyte, bone marrow, mouthwash, lymph node, spleen, liver, biopsy materials, bone. Also, genomic DNA can be successfully obtained from plants.
- DNA can be used in PCR (Polymerase Chain Reaction) reactions and other molecular studies by being isolated from serum and urine samples.
- the present innovative method allows higher quality and quantity of DNA to be obtained compared to current conventional methods by performing DNA isolation processes in a single micro centrifuge tube.
- DNA isolation spread over the liquid phase ensures reliable usage of the method in many areas.
- the product (kit) for application of the present inventive method comprises three solutions (DNA-Y1 , DNA-Y2, DNA-Y3) at sufficient amount and a microcentrifuge tube (DNA-BM) which contains silica matrix, cell lysate, protein and lipid removers at appropriate concentrations for each DNA isolation.
- DNA-BM which is microcentrifuge tube for the present inventive method has a fluid capacity of 2 milliliters in the exemplary embodiment of the invention and provides high-quality DNA isolation from samples (blood or cell suspension for DNA isolation) up to 900 microliters.
- DNA-BM contains DNA binding mix including Tris.HCI (Ph 6.5), EDTA, NaCL, Guanidum Thiocyanate, Nonidet P 40, Tween-20, SDS, DTT, Triton X100, silica beads.
- DNA-Y1 which is the first solution is prepared as an auxiliary solution for purifying DNA from proteins.
- DNA-Y1 contains guanidine thiocyanate that is a chaotic agent, Tris.HCI, Triton X100, EDTA and DTT.
- DNA-Y2 which is the second solution is used for DNA precipitation.
- DNA-Y2 consists of an alcohol mixture containing Tris.HCI, NaCI, Isopropanol and pure Ethanol.
- DNA-Y3 which is the third solution is pure ethyl alcohol. It performs DNA precipitation and alcohol washing processes. Since the preferences of the laboratories may differ, a solution such as distilled water or elution buffer is not provided for DNA elution.
- the process steps occurred in a single tube without using heat, enzyme and high salt concentration from the cells in solution form or from the cells having nucleus in solution form are performed as following way;
- An application example of the present invention is as follows:
- DNA-BM tube mentioned in the method 500 micro liters of blood, bone marrow or cell suspension is transferred to the DNA-BM tube mentioned in the method.
- DNA-BM tube When the present invention is intended to be used for cell- free DNA isolation, DNA is bound by transferring the content of the DNA-BM tube by means of a pipette and similar material onto the desired amount (1-100 milliliters) of the liquid (serum, urine, other body fluids, or other fluids considered to have DNA contamination) from which cell-free DNA is desired to be extracted.
- tubes containing sample biological materials are incubated for 3 minutes at room temperature by mixing (vortex) vigorously. Centrifugation duration is applied as 10 seconds at 10000 rpm for micro tubes.
- the remaining liquid is removed by inverting and tapping it several times on towel paper and similar material.
- the tubes in which, containing samples having cell and cell- free DNA, the DNA-BM content is transferred to, are rotated for 10 minutes at 3500-4000 rpm and supernatant (upper liquid) is carefully removed.
- DNA-Y1 which is the first washing solution
- DNA-BM tubes containing genomic and cell-free DNA 1 milliliter of DNA-Y1 which is the first washing solution, is added on DNA-BM tubes containing genomic and cell-free DNA then, they are mixed and the next step of isolation is quickly put into process. Free DNAs whose cell membranes are broken down and whose membrane, cell lipid and proteins are removed by being disintegrated as a result of the process carried out in both sample tubes, are trapped (bounded) by the silica matrix.
- washing, mixing and centrifugation at 10000 rpm cycle in 10 seconds are performed via DNA-Y1 that is first solution which, being provided with kit, contains guanidine thiocyanate that is a chaotic agent, Tris.HCI, Triton X100, EDTA and DTT. The same process is repeated once again with a second wash with DNA-Y1 content.
- DNA-Y2 that is second solution containing tris base, NaCI, isopropanol and pure Ethanol for precipitation of bounded DNA.
- DNA-BM tubes containing samples with or without cells are inverted and their supernatants are removed.
- DNA precipitation and alcohol washing are performed with DNA-Y3 that is third solution containing pure ethyl alcohol.
- the tubes for the samples with and without cells are dried by allowing the alcohol to evaporate, by leaving their mouths open. Placing them on a heater accelerates the evaporation of alcohol. It can be kept at 65 °C for 10 minutes by adding distilled water in accordance with the sample amounts used for DNA extraction (elution) or adding elution buffer depending on preference and, by mixing them with vortex. Finally, the molten DNAs within the supernatant are taken and spectrophotometric measurements are performed for DNA quality.
- test steps can be stopped at any stage and, it is possible to continue the process in the following days. Increasement in the duration of the incubations increases the amount of DNA obtained. In addition, after removal of the supernatant containing DNA, if required, some more DNA can be obtained by adding elution buffer on the silica matrix.
- DNA isolation is performed quickly and reliably from nucleated cells and fluids containing cell-free DNA.
- the method has application areas for many science and service areas (such as biology, microbiology, immunogenetics, forensics, agriculture, food and veterinary medicine) that require intracellular genomic and cell-free DNA isolation.
- the present innovative kit and method have been developed for HLA tissue typing (with SSP and SSO methods) in the Tissue Typing and Immunology Laboratory. Numerous HLA analyzes were performed with the DNAs isolated by this method. In addition, successful results were obtained in the quantitative PCR studies. The automated DNA isolation device supplied to the laboratory was abandoned due to amplification problem due to low DNA quality and test repeats, and studies were continued with the manual method.
- FIG. 3 another example is the results of Chimerism study in the samples of bone marrow recipient, donor and post-transplant recipient with the DNAs obtained by the present innovative method.
- amelogenin, D5S818, D8S1179, D13S317, D18S52, FAM, HEX, ROX, TAMRA, TPOX, VWA, TH01 genes are successfully amplified with the quantitative PZR method and, comparisons are made.
- D5S818 gene expressions of four recipients and one donor are shown in Figure 3. The clarity of the amplifications can be considered as an indicator of DNA quality.
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Abstract
La présente invention concerne un procédé permettant un isolement d'ADN génomique et libre circulant intracellulaire par l'intermédiaire d'une matrice de silice dans un seul tube sans effectuer les étapes préliminaires du processus de lysat ou d'homogénat de cellules, qu'il est nécessaire d'appliquer avant l'isolement de l'ADN, et un kit associé. Le kit comprend un tube microcentrifuge (BM à l'ADN) qui contient une matrice de silice, des agents d'élimination de lysats de cellules, de protéines et de lipides à des concentrations appropriées pour chaque isolement d'ADN et le BM à l'ADN mentionné contient un mélange de liaison (BM) à l'ADN comprenant du Tris.HCI (à pH 6,5), de l'EDTA, du NaCl, du thiocyanate de guanidium, du Nonidet P 40, du Tween-20, du SDS, du DTT, du Triton X100, des billes de silice.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TR2019/01522 | 2019-02-01 | ||
| TR2019/01522A TR201901522A2 (tr) | 2019-02-01 | 2019-02-01 | Tek tüpte hizli ve yüksek kali̇tede genomi̇k veya hücre dişi dna i̇zolasyonu i̇çi̇n bi̇r yöntem buna i̇li̇şki̇n bi̇r ki̇t |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO2020159461A2 true WO2020159461A2 (fr) | 2020-08-06 |
| WO2020159461A3 WO2020159461A3 (fr) | 2020-09-03 |
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ID=71840579
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/TR2020/050048 WO2020159461A2 (fr) | 2019-02-01 | 2020-01-22 | Procédé d'isolement rapide et de haute qualité d'adn génomique ou libre circulant dans un seul tube et kit associé |
Country Status (2)
| Country | Link |
|---|---|
| TR (1) | TR201901522A2 (fr) |
| WO (1) | WO2020159461A2 (fr) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113462684A (zh) * | 2021-07-30 | 2021-10-01 | 江南大学 | 一种基于纸芯片快速提取超纯dna的方法 |
| CN114517196A (zh) * | 2022-02-25 | 2022-05-20 | 杭州诺辉健康科技有限公司 | 一种血浆游离miRNA提取方法及其应用 |
| CN117126843A (zh) * | 2023-09-18 | 2023-11-28 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | 一种用于小型浮游动物单个体的dna提取方法 |
| CN118389492A (zh) * | 2024-07-01 | 2024-07-26 | 广州凯普医药科技有限公司 | 一种磁珠法提取干血斑基因组dna的试剂盒及其应用 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5234809A (en) * | 1989-03-23 | 1993-08-10 | Akzo N.V. | Process for isolating nucleic acid |
| EP2128169A1 (fr) * | 2008-05-30 | 2009-12-02 | Qiagen GmbH | Procédé d'isolation d'acides nucléiques à chaînes courtes |
-
2019
- 2019-02-01 TR TR2019/01522A patent/TR201901522A2/tr unknown
-
2020
- 2020-01-22 WO PCT/TR2020/050048 patent/WO2020159461A2/fr active Application Filing
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113462684A (zh) * | 2021-07-30 | 2021-10-01 | 江南大学 | 一种基于纸芯片快速提取超纯dna的方法 |
| CN113462684B (zh) * | 2021-07-30 | 2023-09-05 | 江南大学 | 一种基于纸芯片快速提取超纯dna的方法 |
| CN114517196A (zh) * | 2022-02-25 | 2022-05-20 | 杭州诺辉健康科技有限公司 | 一种血浆游离miRNA提取方法及其应用 |
| CN117126843A (zh) * | 2023-09-18 | 2023-11-28 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | 一种用于小型浮游动物单个体的dna提取方法 |
| CN117126843B (zh) * | 2023-09-18 | 2024-05-14 | 生态环境部华南环境科学研究所(生态环境部生态环境应急研究所) | 一种用于小型浮游动物单个体的dna提取方法 |
| CN118389492A (zh) * | 2024-07-01 | 2024-07-26 | 广州凯普医药科技有限公司 | 一种磁珠法提取干血斑基因组dna的试剂盒及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2020159461A3 (fr) | 2020-09-03 |
| TR201901522A2 (tr) | 2020-08-21 |
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