In vitro sorting method

• US 6,489,103 B1
• Filed: 12/16/1999
• Issued: 12/03/2002
• Est. Priority Date: 07/07/1997
• Status: Expired due to Term

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Griffiths, Andrew, Tawfik, Dan
• Primary Examiner(s): Reynolds, Deborah J.
• Assistant Examiner(s): Sorbello, Eleanor
• Application Number:
  US09/464,122
• Time in Patent Office: 1,083 Days
• Field of Search: 424/501, 424/426, 435/5, 435/320.1, 435/69.1, 514/44, 536/23.1
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components

Abstract

The invention relates to 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 a desired activity. The invention permits the in vitro evolution of nucleic acids by repeated mutagenesis and iterative applications of the method of the invention.

Claims

1. 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 said genetic elements to produce, within said microcapsules, gene products encoded by said genetic elements; and

(c) sorting said genetic elements which produce a gene product or products having the desired activity.

2. The method according to claim 1 wherein said gene product with the desired activity induces a change in the microcapsule which enables the microcapsule containing the gene product and the genetic element encoding it to be sorted.

3. The method according to claim 2 wherein said gene product having the desired activity modifies one or more molecules within the microcapsule which enables the microcapsule containing the gene product and the genetic element encoding it to be sorted.

4. The method according to claim 2 or claim 3 wherein the gene product having the desired activity induces a change in the optical properties of the microcapsule wherein said change permits the microcapsule to be sorted.

5. The method according to claim 1, wherein the desired activity of said gene product within said microcapsule results, directly or indirectly, in the modification of said genetic element encoding said gene product to enable the isolation of said genetic element.

6. The method according to claim 5 wherein the gene product having the desired activity induces a change in the microcapsule which, when detected, triggers the modification of the genetic element within the compartment to enable the isolation of the genetic element.

7. The method according to claim 6 wherein the change in the microcapsule is a change in the optical properties thereof.

8. The method according to claim 5 wherein said genetic element comprises a substrate and the activity of the desired gene product within the microcapsule results in the conversion of said substrate into a product which remains linked to the genetic element and enables its isolation.

9. The method according to claim 5 wherein said genetic element comprises a ligand and the desired gene product within the microcapsule binds to said ligand to enable the isolation of the genetic element.

10. The method according to claim 1 wherein the activity of said gene product within said microcapsule results in the alteration of the expression of a second gene within the compartment and the activity of the product of said second gene enables the isolation of said genetic element.

11. The method according to claim 1, wherein step (b) comprises:

expressing the genetic elements to produce their respective gene products within the microcapsules, linking the gene products to the genetic elements encoding them and isolating the complexes thereby formed.

12. The method according to claim 11, wherein the complexes are subjected to a further compartmentalisation step in order to isolate the genetic elements encoding a gene product having the desired activity.

13. The method according to claim 12 wherein the gene product having the desired activity permits the microcapsule containing the gene product to be sorted.

14. The method according to claim 11 wherein step (c) comprises isolating the genetic element as a result of the activity of the desired gene product within the microcapsule.

15. The method of claim 14 wherein the gene product having the desired activity induces a change in the microcapsule which triggers the modification of the genetic element.

16. The method of claim 15 wherein the modification of the genetic element permits the isolation of the genetic element.

17. The method of claim 15 wherein the change in the microcapsule is a change in the optical properties thereof.

18. The method of claim 14 wherein the genetic element comprises a substrate and the activity of the desired gene product within the microcapsule results in the conversion of said substrate into a product which remains linked to the genetic element and permits its isolation.

19. The method of claim 14 wherein the genetic element is a ligand and the desired gene product within the microcapsule binds to said ligand to permit the isolation of the genetic element.

20. The method of claim 14 wherein the activity of the desired gene product within the microcapsule permits altered expression of a second gene within the microcapsule and the activity of the product of the second gene permits the isolation of the genetic element.

21. The method according to claim 1, wherein said genetic elements are isolated from a library of genetic elements encoding a repertoire of gene products.

22. The method according to claim 1, further comprising the step of:

(d) introducing one or more mutations into said genetic element or elements sorted in step (c).

23. The method according to claim 22, further comprising iteratively repeating at least one of steps (a) to (d).

24. The method according to claim 1, further comprising amplifying said genetic elements.

25. The method of claim 1 wherein a reportoire of genetic elements encoding a respective repertoire of gene products is isolated by:

(a) compartmentalizing the elements into microcapsules;

(b) expressing the genetic elements to produce their respective gene products; and

(c) sorting the genetic elements.

26. The method according to claim 25, wherein a library of genetic elements encoding a repertoire of gene products is encapsulated and each genetic element expressed to form its gene product within a microcapsule.

27. The method according to claim 25 or claim 26, wherein microencapsulation is achieved by forming a water-in-oil emulsion of the aqueous solution in an oil-based medium.

28. The method according to claim 1, wherein in step (c), the genetic elements are sorted by affinity purification.

29. The method according to claim 1, wherein in step (c), the genetic elements are sorted by selective ablation of genetic elements which do not encode the desired gene product.

30. A method for preparing a gene product, comprising the steps of:

(a) preparing a genetic element encoding said gene product;

(b) compartmentalising said genetic element into a microcapsule;

(c) expressing said genetic element to produce, within the microcapsules, the gene product encoded by said genetic element;

(d) sorting said genetic element which produces said gene product; and

(e) expressing said gene product.