In vitro sorting method

• US 20050069920A1
• Filed: 06/03/2004
• Published: 03/31/2005
• Est. Priority Date: 07/07/1997
• Status: Active Grant

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Griffiths, Andrew, Tawfik, Dan
• Granted Patent: US 7,252,943 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising of 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 enables the in vitro evolution of nucleic acids by repeated mutagenesis and iterative applications of the method of the invention.

Claims

1. Cancelled

2. A method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising the steps of:

(a) expressing the genetic elements to produce their respective gene products such that the gene products are linked to the genes encoding them;

(b) compartmentalising the genetic elements into microcapsules;

(c) sorting the genetic elements which produce gene product(s) having the desired activity.

3. The method according to claim 2 wherein a 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.

4. The method according to claim 3 wherein a 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.

5. The method according to claim 3 wherein the gene product having the desired activity induces a change in the optical properties of the microcapsule and the microcapsules are sorted thereby.

6. The method according to claim 5 wherein the change in optical properties is a change in fluorescence.

7. The method according to claim 2, wherein the desired activity of the gene product within the microcapsule results, directly or indirectly, in the modification of the genetic element encoding the gene product to enable the isolation of the genetic element.

8. The method according to claim 7 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.

9. The method according to claim 8 wherein the change in the microcapsule is a change in the optical properties thereof.

10. The method according to claim 9, wherein the change in optical properties is a change in fluorescence.

11. The method according to claim 7 wherein a part of the genetic element is a substrate and the activity of the desired gene product within the microcapsule results, directly or indirectly, in the conversion of said substrate into a product which remains part of the genetic element and enables its isolation.

12. The method according to claim 7 wherein the activity of the desired gene product within the microcapsule results, directly or indirectly, in the conversion of a substrate into a product, and the genetic element further comprises a ligand which binds to said product and enables its isolation.

13. The method according to claim 7 wherein the 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.

14. The method according to claim 2 wherein the activity of the gene product within the microcapsule results in the alteration of the expression of a second gene within the compartment and the activity of the product of the said second gene enables the isolation of the genetic element

15. The method according to claim 2, wherein the genetic elements are isolated from a library of genetic elements encoding a repertoire of gene products.

16. The method according to claim 2 further comprising the additional step of:

(d) introducing one or more mutations into the genetic element(s) isolated in step (c).

17. The method according to claim 16 further comprising iteratively repeating one or more of steps (a) to (d).

18. The method according to claim 2 further comprising amplifying the genetic elements.

19. The method according to claim 2, wherein microencapsulation is achieved by forming a water-in-oil emulsion of an aqueous solution in an oil-based medium.

20. The method according to claim 2, wherein the genetic elements are sorted by affinity purification.

21. The method according to claim 2, wherein the genetic elements are sorted by selective ablation of genetic elements which do not encode the desired gene product.

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

(d) preparing a genetic clement encoding the gene product;

(e) expressing the genetic elements to produce their respective gene products such that the gene products are linked to the genetic elements encoding them;

(f) compartmentalising genetic elements into microcapsules;

(g) sorting the genetic elements which produce the gene product(s) having the desired activity; and

(h) expressing the gene product having the desired activity.

23. A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of:

(i) preparing a genetic element encoding the gene product;

(j) expressing the genetic elements to produce their respective gene products and linking the gene products to the genetic elements encoding them;

(k) compartmentalising genetic elements into microcapsules;

(l) sorting the genetic elements which produce the gene product(s) having the desired activity; and

(m) 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.

24. A method for preparing a compound or compounds comprising the steps of:

(n) providing a synthesis protocol wherein at least one step is facilitated by a polypeptide;

(o) preparing genetic elements encoding variants of the polypeptide which facilitates this step;

(p) expressing the genetic elements to produce their respective gene products such that the gene products are linked to the genetic elements encoding them;

(q) compartmentalising the genetic elements into microcapsules;

(r) sorting the genetic elements which produce polypeptide gene product(s) having the desired activity and

(s) preparing the compound or compounds using the polypeptide gene product identified in (e) to facilitate the relevant step of the synthesis.