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Kafe 바로가기국가/구분 | United States(US) Patent 등록 |
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국제특허분류(IPC7판) |
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출원번호 | US-0281247 (2005-11-16) |
등록번호 | US-8852862 (2014-10-07) |
발명자 / 주소 |
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출원인 / 주소 |
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대리인 / 주소 |
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인용정보 | 피인용 횟수 : 32 인용 특허 : 602 |
Methods and systems for processing polynucleotides (e.g., DNA) are disclosed. A processing region includes one or more surfaces (e.g., particle surfaces) modified with ligands that retain polynucleotides under a first set of conditions (e.g., temperature and pH) and release the polynucleotides under
Methods and systems for processing polynucleotides (e.g., DNA) are disclosed. A processing region includes one or more surfaces (e.g., particle surfaces) modified with ligands that retain polynucleotides under a first set of conditions (e.g., temperature and pH) and release the polynucleotides under a second set of conditions (e.g., higher temperature and/or more basic pH). The processing region can be used to, for example, concentrate polynucleotides of a sample and/or separate inhibitors of amplification reactions from the polynucleotides. Microfluidic devices with a processing region are disclosed.
1. A method for separating one or more polynucleotides from a sample containing polymerase chain reaction inhibitors, the method comprising: contacting a solution of the sample with a plurality of polynucleotide binding particles, wherein the binding particles are configured to preferentially retain
1. A method for separating one or more polynucleotides from a sample containing polymerase chain reaction inhibitors, the method comprising: contacting a solution of the sample with a plurality of polynucleotide binding particles, wherein the binding particles are configured to preferentially retain the one or more polynucleotides in the sample as compared to polymerase chain reaction inhibitors;wherein the sample solution has a volume from 0.5 microliters to 3 milliliters;wherein the plurality of binding particles have a volume less than 5 microliters, and surfaces that comprise a polycationic polyamide configured to bind polynucleotides in preference to polymerase chain reaction inhibitors at a pH of 8.5 or less;removing the solution containing inhibitors from the plurality of binding particles; andreleasing the one or more polynucleotides from the binding particles into a single volume of liquid wherein the ratio of the volume of sample solution to the volume of liquid into which the polynucleotides are released is between 50:1 and 1000:1, wherein the releasing occurs at a pH of 11.4 or greater. 2. The method of claim 1, wherein the one or more polynucleotides has a size of less than 7.5 Mbp. 3. The method of claim 1, wherein contacting the sample with the plurality of polynucleotide binding particles comprises actuating a thermally actuated pressure source to apply a pressure to the sample. 4. The method of claim 3, wherein contacting the sample with the plurality of polynucleotide binding particles comprises opening a thermally actuated valve to place the sample in fluid communication with the binding particles. 5. The method of claim 1, additionally comprising, prior to the releasing step, washing the particles with a volume of wash solution less than 50 microliters. 6. The method of claim 5 wherein the wash solution comprises a detergent. 7. The method of claim 1, wherein the polymerase chain reaction inhibitors comprise at least one of hemoglobin, peptides, faecal compounds, humic acids, mucousol compounds, DNA binding proteins, or a saccharide. 8. The method of claim 1, wherein the polycationic polyamide is polyethyleneimine. 9. The method of claim 8, wherein the polyethyleneimine has a molecular weight in the range 600-800 Da. 10. The method of claim 1, wherein the polycationic polyamide is selected from the group consisting of poly-DL-ornithine, poly-L-lysine, and poly-D-lysine. 11. The method of claim 1, wherein the releasing comprises heating the plurality of binding particles to a temperature of between about 50° C. and about 100° C. 12. The method of claim 11, wherein the plurality of binding particles is heated in the presence of a liquid and the temperature is insufficient to boil the liquid in the presence of the plurality of binding particles during heating. 13. The method of claim 11, wherein the temperature is maintained for between about 1 and 10 minutes. 14. The method of claim 1, wherein the method does not comprise centrifugation of the binding particles. 15. The method of claim 1, wherein the time required for completing the contacting, concentrating, and releasing steps is less than 15 minutes. 16. The method of claim 1, wherein the sample has a volume larger than the volume of the polynucleotide binding particles having the one or more polynucleotides bound thereto by a factor of at least about 10. 17. The method of claim 1, wherein the polycationic polyamide is covalently bound to the surfaces of the binding particles. 18. The method of claim 1, wherein the polycationic polyamide is poly-L-lysine or poly-D-lysine and has an average molecular weight of between about 7,500 Da and about 35,000 Da. 19. The method of claim 1, wherein the polycationic polyamide is poly-L-lysine or poly-D-lysine and has a median molecular weight of between about 15,000 Da and about 25,000 Da. 20. The method of claim 1, wherein the binding particles are made of a polymeric material selected from the group consisting of: polystyrene, latex polymers, polyacrylamide, and polyethylene oxide. 21. The method of claim 20, wherein the polymeric material is modified to provide one or more carboxylic groups and/or one or more amino groups, wherein the groups provide an attachment point for one or more ligands. 22. The method of claim 1, wherein the binding particles have an average diameter of between about 4 microns and about 20 microns. 23. The method of claim 1, wherein the binding particles are present in a density of about 108 particles per milliliter. 24. The method of claim 1, wherein at least some of the binding particles are magnetic. 25. The method of claim 1, wherein the polycationic polyamide is resistant to pronase degradation. 26. The method of claim 1, wherein the contacting takes place in the presence of lysis reagents. 27. The method of claim 26, wherein the contacting in the presence of lysis reagents comprises heating the sample to a temperature more than 50° C. 28. The method of claim 26, wherein the contacting in the presence of lysis reagents takes place for a period of time between 5 to 15 minutes.
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