Optical sorting method
First Claim
1. A method for isolating one or more genetic elements encoding a gene product having a desired activity the expression of which results, directly or indirectly, in the modification of an optical property of a genetic element encoding the gene product, comprising the steps of:
- (a) compartmentalising genetic elements into microcapsules;
(b) expressing the genetic elements to produce their respective gene products within the microcapsules;
(c) sorting the genetic elements which produce the gene product(s) having the desired activity according to a change in the optical properties of the genetic elements, whereby a genetic element encoding a gene product having said desired activity is isolated.
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Abstract
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) compartmentalising genetic elements into microcapsules; (b) expressing the genetic elements to produce their respective gene products within the microcapsules; (c) sorting the genetic elements which produce the gene product having the desired activity using a change in the optical properties of the genetic elements. The invention enables the in vitro evolution of nucleic acids and proteins by repeated mutagenesis and iterative applications of the method of the invention.
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Citations
43 Claims
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1. A method for isolating one or more genetic elements encoding a gene product having a desired activity the expression of which results, directly or indirectly, in the modification of an optical property of a genetic element encoding the gene product, comprising the steps of:
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(a) compartmentalising genetic elements into microcapsules;
(b) expressing the genetic elements to produce their respective gene products within the microcapsules;
(c) sorting the genetic elements which produce the gene product(s) having the desired activity according to a change in the optical properties of the genetic elements, whereby a genetic element encoding a gene product having said desired activity is isolated. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
(d) introducing one or more mutations into the genetic element(s) isolated in step (c).
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31. The method according to claim 1, said method further comprising iteratively repeating one or more of steps (a) to (d).
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32. The method according to claim 1, said method further comprising amplifying the genetic elements.
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33. The method according to claim 1, wherein said compartmentalizing comprises microencapsulation achieved by forming a water-in-oil emulsion of an aqueous solution in an oil-based medium.
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34. The method according to claim 1, wherein said one or more the genetic elements comprise a gene attached to a microbead.
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35. The method according to claim 34, wherein the microbead is nonmagnetic, magnetic or paramagnetic.
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36. The method according to claim 1, wherein said sorting is based on a change in fluorescence of a genetic element encoding a gene product having said desired activity.
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37. The method according to claim 36, wherein said sorting is performed using a fluorescence activated cell sorter (FACS) or its equivalent.
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38. The method according to claim 36, wherein said change in fluorescence is due to fluorescence resonance energy transfer (FRET).
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39. The method according to claim 1, wherein the internal environment of said microcapsules is modified by the addition of one or more reagents to the oil phase.
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40. The method according to claim 1, wherein genetic elements modified directly or indirectly by the activity of the desired gene product are further modified by tyramide signal amplification, resulting directly or indirectly in a change in the optical properties of said genetic elements thereby enabling their separation.
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41. A method for preparing a gene product, comprising the steps of:
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(a) preparing plurality of a genetic elements encoding a plurality of gene products;
(b) compartmentalising said genetic elements into microcapsules;
(c) expressing the genetic elements to produce their respective gene products within the microcapsules;
(d) sorting the genetic elements which produce the gene product(s) having the desired activity using a change in their optical properties; and
(e) expressing the gene product having the desired activity.
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42. A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of:
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(a) preparing a repertoire of genetic elements encoding gene products;
(b) compartmentalising the genetic elements into microcapsules;
(c) expressing the genetic elements to produce their respective gene products within the microcapsules;
(d) sorting the genetic elements which produce the gene product(s) having the desired activity using a change in their optical properties; and
(e) contacting a gene product having the desired activity with the compound or compounds and monitoring the modulation of an activity of the gene product by the compound or compounds.
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43. In a method of preparing a compound, the method comprising a step of contacting a polypeptide with a reagent, wherein said contacting facilitates at least one step in said method of preparing a compound, the improvement comprising:
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a) preparing genetic elements encoding variants of said polypeptide;
b) compartmentalizing said genetic elements into microcapsules;
c) expressing the genetic elements to produce their respective gene products within the microcapsules;
d) sorting a genetic element which produces a polypeptide gene product having a desired activity using a change in an optical property of the genetic element to sort, wherein a gene product having said desired activity is identified; and
e) using the polypeptide identified in step (d) in said method of preparing a compound.
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Specification