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1. WO2020159461 - PROCÉDÉ D'ISOLEMENT RAPIDE ET DE HAUTE QUALITÉ D'ADN GÉNOMIQUE OU LIBRE CIRCULANT DANS UN SEUL TUBE ET KIT ASSOCIÉ

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CLAIMS

1. A DNA isolation kit for a method allowing intracellular genomic and cell-free DNA isolation via silica matrix in a single tube without performing the preliminary steps of the cell lysate or homogenate process, that are required to be applied prior to DNA isolation, and characterized by;

comprising a microcentrifuge tube (DNA-BM) that contains silica matrix, cell lysate, protein and lipid removers at appropriate concentrations for each DNA isolation and,

mentioned 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.

2. A DNA isolation kit according to Claim 1 and, characterized by; comprising DNA-Y1 solution which is prepared as an auxiliary solution for purifying DNA from proteins and,

mentioned DNA-Y1 contains guanidine thiocyanate that is a chaotic agent, Tris.HCI, Triton X100, EDTA and DTT.

3. A DNA isolation kit according Claim 1 and, characterized by comprising DNA-Y2 solution which is used for DNA precipitation and,

mentioned DNA-Y2 consists of an alcohol mixture containing Tris.HCI, NaCI, Isopropanol and pure Ethanol.

4. A DNA isolation kit according Claim 1 and, characterized by comprising DNA-Y3 solution which is used for performing DNA precipitation and alcohol washing processes and,

mentioned DNA-Y3 contains pure ethyl alcohol.

5. A DNA isolation method allowing intracellular genomic and cell-free DNA isolation via silica matrix in a single tube without performing the preliminary steps of the cell lysate or homogenate process, that are required to be applied prior to DNA isolation, and characterized by;

fragmentation of membranes, hemoglobin and other proteins with a microcentrifuge tube (DNA-BM) that contains silica matrix, cell lysate, protein and lipid removers at appropriate concentrations for each DNA isolation and, binding of DNA with untreated silica matrix, removal of proteins and lipids from the environment by washing processes via DNA-Y1 that is prepared as an auxiliary solution for purifying DNA from proteins,

precipitation of DNA via an DNA-Y2 solution, finally performing both DNA precipitation and alcohol washing via a DNA-Y3 solution.

6. A DNA isolation method according to Claim 5 and, wherein mentioned DNA-BM is a DNA binding mix obtained by mixing Tris.HCI (Ph 6.5), EDTA, NaCL, Guanidum Thiocyanate, Nonidet P 40, Tween-20, SDS, DTT, Triton X100, silica beads.

7. A DNA isolation method according to Claim 5 and, wherein mentioned DNA-Y1 is obtained by mixing guanidine thiocyanate that is a chaotic agent, Tris.HCI, Triton X100, EDTA and DTT.

8. A DNA isolation method according to Claim 5 and, wherein mentioned DNA-Y2 is obtained by mixing an alcohol mixture containing Tris.HCI, NaCI, Isopropanol and pure Ethanol.

9. A DNA isolation method according to Claim 5 and, wherein mentioned DNA-Y3 comprises pure ethyl alcohol.

10. A DNA isolation method according to one of the preceding claims and, characterized by;

transferring said DNA-BM tube content into the tube containing liquid in which DNA is desired to be isolated, for DNA isolation,

vortexing and incubating the tube containing DNA sample and DNA-BM content at room temperature,

removing the remaining liquid from over the tube containing said DNA sample, transferring of DNA sample into DNA-BM or other microcentrifuge tube after removal of remaining liquid,

performing washing, mixing and centrifugation processes by adding said DNA-Y1 tube content which is first solution containing guanidine thiocyanate that is a chaotic agent, Tris, Triton X100, EDTA and DTT, onto mentioned tube,

repeating the washing, mixing and centrifugation processes by performing a second wash with the content of DNA-Y1 tube content,

performing of the washing operation with the DNA-Y2 tube content which is second solution containing tris base, NaCI, isopropanol and pure ethanol, for precipitating the bounded DNA,

removing of the supernatants by inverting the tube at the end of the centrifugation process,

performing alcohol washing with DNA-Y3 which is the third solution containing pure ethyl alcohol for DNA precipitation and alcohol washing,

repeating of the centrifugation process again and, after removal of the supernatant, leaving the mouth of the tube open and ensuring the alcohol to evaporate,

- placing of elution buffer or distilled water in accordance with the sample amounts used for DNA extraction (elution), mixing them with vortex and, taking the molten DNA from the supernatant and, making the measurements.