In vitro sorting method

• US 9,528,106 B2
• Filed: 01/03/2013
• Issued: 12/27/2016
• 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): Babic, Christopher M
• Assistant Examiner(s): Hiriyanna, Kelaginamane T
• Application Number:
  US13/733,473
• Publication Number:
  US 20130190189A1
• Time in Patent Office: 1,454 Days
• Field of Search: 435/6.1
• US Class Current: 1/1
• 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) compartmentalizing 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 isolating one or more genetic elements encoding a gene product having a desired catalytic activity, comprising the steps of:

(a) compartmentalising the genetic elements into microcapsules;

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

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

wherein the desired catalytic activity of the gene product results, directly or indirectly, in the enzymatic modification of the genetic element encoding the gene product or the microcapsule containing it to enable the isolation of the genetic element, and wherein the gene product having the desired catalytic 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.

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

(a) compartmentalising the genetic elements into microcapsules;

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

(c) sorting the genetic elements which produce gene product(s) having the desired catalytic activity, wherein the genetic element encodes a dihydrofolate reductase gene.

3. A method according to claim 2, wherein the genetic element encodes a bacterial dihydrofolate reductase gene.

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

(a) compartmentalising the genetic elements into microcapsules;

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

(c) sorting the genetic elements which produce gene product(s) having the desired catalytic activity, wherein the genetic element is linked to a plurality of substrate molecules.

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

(a) compartmentalising the genetic elements into microcapsules;

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

(c) sorting the genetic elements which produce gene product(s) having the desired catalytic activity, wherein the isolating is by affinity purification of said genetic elements.

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

(a) compartmentalising the genetic elements into microcapsules;

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

(c) sorting the genetic elements which produce gene product(s) having the desired catalytic activity, wherein the isolating is by affinity purification using an antibody that binds specifically to an antigen in the gene product.

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

(a) compartmentalising the genetic elements into microcapsules;

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

(c) sorting the genetic elements which produce gene product(s) having the desired catalytic activity, wherein the microcapsules are formed from an emulsion comprising a heterogenous system of a first immiscible liquid phase and a second immiscible liquid phase, wherein the first immiscible liquid phase is dispersed in the second immiscible liquid phase as droplets of microscopic or colloidal size.

8. A method according to claim 7, wherein the emulsion has water as the first immiscible liquid phase present in the form of finely divided droplets and the second immiscible liquid phase is a hydrophobic, immiscible liquid phase.

9. A method according to claim 7, wherein said heterogeneous system is a water-in-oil emulsion.

10. A method according to claim 9, wherein said emulsion further comprises at least one surfactant.

11. A method according to claim 10, wherein said at least one surfactant is an anionic surfactant.

12. A method according to claim 11, wherein said at least one surfactant is selected from the group consisting of sodium cholate and sodium taurocholate.