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Kafe 바로가기국가/구분 | United States(US) Patent 등록 |
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국제특허분류(IPC7판) |
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출원번호 | US-0692615 (2012-12-03) |
등록번호 | US-9186643 (2015-11-17) |
발명자 / 주소 |
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출원인 / 주소 |
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대리인 / 주소 |
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인용정보 | 피인용 횟수 : 0 인용 특허 : 421 |
The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalizing genetic elements into microcapsules; and (b) sorting the genetic elements which express the gene product having the desired a
The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of: (a) compartmentalizing genetic elements into microcapsules; and (b) sorting the genetic elements which express the gene product having the desired activity; wherein at least one step is under microfluidic control. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.
1. A method for producing an encapsulated gene product, comprising the steps of: (a) providing within a first microcapsule within a microfluidic channel a first genetic element encoding a first gene product, the microcapsule comprising an aqueous fluid substantially surrounded by an immiscible fluor
1. A method for producing an encapsulated gene product, comprising the steps of: (a) providing within a first microcapsule within a microfluidic channel a first genetic element encoding a first gene product, the microcapsule comprising an aqueous fluid substantially surrounded by an immiscible fluorinated oil including a fluorinated polymer surfactant, wherein the microcapsule is able to contact without fusing with another microcapsule due to presence of the surfactant;(b) providing conditions within the first microcapsule suitable for the expression of the first genetic element to form its first gene product;(c) introducing at least one of a first microbead to the first microcapsule, wherein the first microbead is either encapsulated within the first microcapsule prior to the expression of the first genetic element or is encapsulated within the first microcapsule subsequent to the expression of the first genetic element; and(d) complexing the first gene product with at least one of the plurality of first microbeads via a capture moiety on the first microbead. 2. The method of claim 1, further comprising the step of (e) detecting a selectable change caused by the first genetic element. 3. The method of claim 2, wherein the first genetic element indirectly causes the selectable change by the first gene product complexing with the first microbead. 4. The method of claim 3, wherein the first gene product comprises an antibody and the selectable change is caused, directly or indirectly, by the antibody binding to an antigen directly or indirectly attached to the first microbead. 5. The method of claim 2, wherein the first genetic element indirectly via the first gene product causes the selectable change to a complex linked to the first microbead. 6. The method of claim 2, further comprising the step of (f) sorting the first microcapsule based on detecting the selectable change. 7. The method of claim 6, further comprising the step of (g) cloning the first genetic element to produce recombinant protein. 8. The method of claim 6, wherein the sorting is done using a fluorescence activated cell sorter (FACS) or similar device. 9. The method of claim 2, wherein the selectable change comprises an optical signal. 10. The method of claim 1, wherein the first genetic element is contained within a cell. 11. The method of claim 10, wherein the cell is an immune cell. 12. The method of claim 11, wherein the first gene product is a protein. 13. The method of claim 12, wherein the protein is an antibody. 14. The method of claim 12, wherein the protein is located within the cell and/or outside of the cell within the microcapsule. 15. The method of claim 10, wherein the cell produces a gene product that has enzymatic or binding activity. 16. The method of claim 1, wherein the one or more microbeads are provided within the first microcapsule when the first microcapsule is formed. 17. The method of claim 1, wherein the one or more microbeads are encapsulated in a second microcapsule, and further comprising the step of merging the first microcapsule with the second microcapsule to provide the one or more microbeads in the first microcapsules. 18. The method of claim 1, wherein the expression comprises amplifying the gene element. 19. The method of claim 18, wherein the first genetic element is DNA and wherein step (c), if the expression occurs, comprises increasing the concentration of the first genetic element by amplification. 20. The method of claim 19, wherein the amplification is the polymerase chain reaction (PCR). 21. The method of claim 19, wherein the product of the expression is complexed to the first microbead. 22. The method of claim 21, wherein the product of the expression is a PCR product. 23. The method of claim 21, wherein the first microcapsule comprises the first genetic element and further comprising the steps of: (e) providing a plurality of second microbeads;(f) providing a plurality of second microcapsules each comprising at least one of the plurality of second microbeads and a number of genetic elements encoding gene products, wherein the number is determined by a distribution such that a majority of the second microcapsules each contain no more than one gene element per droplet; and(g) repeating steps (a) through (d) for each of the second microcapsules, wherein step (c), if the expression occurs, comprises increasing the concentration of the genetic elements in the second microcapsules by amplification and wherein the product of the expression (expression product) in each of the second microcapsules is complexed to the at least one second microbead via a capture moiety on the second microbead. 24. The method of claim 23, further comprising the step of (h) pooling the first microbead and the second microbeads, optionally further comprising the step of(i) sequencing the expression products complexed to one or more of the pooled microbeads. 25. The method of claim 23, further comprising the step of (h) sorting the second microcapsules to isolate those of the second microcapsules wherein the expression occurred, optionally further comprising the step of(i) pooling the first microbead and the isolated microbeads, preferably further comprising the step of(j) sequencing the expression products complexed to one or more of the pooled microbeads. 26. The method of claim 1, wherein the first microcapsule comprises either no genetic elements or the first genetic element and no other genetic elements. 27. The method of claim 1, further comprising performing steps (b) through (d) on a second microcapsule within the microfluidic channel wherein the second microcapsule comprises a second genetic element encoding a second gene product; and further comprising the step of (e) pooling the first and second microcapsules. 28. The method of claim 1, wherein the conditions include providing a substrate. 29. The method of claim 1, wherein the first genetic element is DNA and its expression comprises conversion of the DNA into protein. 30. The method of claim 1, wherein the first microbead is linked to the first genetic element. 31. The method of claim 1, further comprising the step of providing a plurality of microcapsules that are highly monodisperse with the first microcapsule. 32. The method of claim 1, wherein step (a) further comprises providing within the first microcapsule a first cell that is the single cell in the first microcapsule and wherein the first genetic element is contained within the first cell, and wherein step (b) further comprises providing that the first microcapsule contains a first microbead that is the single microbead in the first microcapsule. 33. The method of claim 32, wherein the first genetic element is DNA and wherein step (b), if the expression occurs, comprises increasing the concentration of the first genetic element by amplification. 34. The method of claim 32, further comprising the steps of: (e) providing a plurality of second microcapsules, each comprising a single one of the microbeads and each further comprising a single cell containing a single one of a plurality of second genetic elements; and(f) repeating steps (a) through (d) for each of the second microcapsules, wherein step (c), if the expression occurs, comprises increasing the concentration of the respective single genetic elements in the first and second microcapsules by amplification and wherein the product of the expression (expression product) in each of the first and second microcapsules is complexed to the single microbead in the respective microcapsule via a capture moiety on the single microbead. 35. The method of claim 34, further comprising the step of (g) pooling the first microbead and the second microbeads. 36. The method of claim 32, wherein the first genetic element is DNA in the first cell and the first gene product is RNA complexed to the first microbead. 37. The method of claim 32, wherein the first genetic element is RNA and the first gene product is cDNA complexed to the first microbead. 38. The method of claim 1, wherein the introducing comprises fusing the first microcapsule with a microcapsule that comprises the first microbead in the presence of an electric field.
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