In vitro sorting method

• US 8,367,326 B2
• Filed: 06/21/2007
• Issued: 02/05/2013
• Est. Priority Date: 07/07/1997
• Status: Expired due to Fees

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Griffiths, Andrew, Tawfik, Dan
• Primary Examiner(s): Kelly, Robert M
• Assistant Examiner(s): Hiriyanna, Kelaginamane T
• Application Number:
  US11/820,903
• Publication Number:
  US 20070259374A1
• Time in Patent Office: 2,056 Days
• Field of Search: None
• US Class Current: 435/6.1
• 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. A method for screening a compound or compounds capable of modulating the activity of a gene product, comprising the steps of:

(a) preparing a repertoire of genetic elements encoding the gene product;

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

(c) compartmentalizing the genetic elements into microcapsules;

(d) sorting the genetic elements which produce the gene product(s) having a desired activity; 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.

2. The method according to claim 1 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.

3. The method according to claim 2 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.

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 and the microcapsules are sorted thereby.

5. The method according to claim 1, 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.

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 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.

9. The method according to claim 5 wherein a part of the genetic element is a ligand and the desired gene product within the microcapsule binds, directly or indirectly, to said ligand to enable the isolation of the genetic element.

10. The method of claim 1, wherein the activity of the desired gene product within the microcapsule results, directly or indirectly, 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.

11. The method of claim 1, wherein the genetic elements are isolated from a library of genetic elements encoding a repertoire of gene products.

12. The method of claim 1, further comprising the additional step of:

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

13. The method of claim 12, further comprising iteratively repeating one or more of steps (a) to (f).

14. The method of claim 1, further comprising amplifying the genetic elements.