In vitro sorting method

• US 7,582,446 B2
• Filed: 09/08/2006
• Issued: 09/01/2009
• 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): VOGEL, NANCY TREPTOW
• Application Number:
  US11/518,094
• Publication Number:
  US 20070077579A1
• Time in Patent Office: 1,089 Days
• Field of Search: None
• US Class Current: 435/69.1
• 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 of preparing a gene product, said method comprising:

a) expressing a genetic element of a plurality of genetic elements to produce an expressed product, wherein said plurality of said genetic elements are dispersed in aqueous droplets of an emulsion;

b) separating an aqueous droplet that contains said expressed product from those that do not; and

c) further expressing the genetic element present in said aqueous droplet, thereby producing said gene product.

2. The method of claim 1, wherein said emulsion is a water-in-oil emulsion.

3. The method of claim 1, wherein a plurality of droplets comprise a single genetic element.

4. The method of claim 1, wherein the aqueous phase of said emulsion further comprises a molecule that interacts with a said expressed product to generate a detectable signal.

5. The method of claim 4, wherein said separating step comprises detection of said detectable signal.

6. The method of claim 4, wherein said molecule is a substrate of a said expressed product.

7. The method of claim 4, wherein detection comprises detecting a change in an optical property of a droplet.

8. The method of claim 7, wherein said optical property is fluorescence.

9. The method of claim 1, wherein said step of separating comprises fluorescence activated sorting of said droplets.

10. The method of claim 1, wherein said plurality of genetic elements is a library of genetic elements.

11. The method of claim 1, wherein said plurality of genetic elements are physically associated with a known ligand, and said step of separating comprises separating a genetic element that encodes a product that binds said ligand from those that do not.

12. The method of claim 11, wherein said genetic elements each comprise a sequence encoding a peptide or polypeptide tag, and step (b) comprises the steps of:

i) pooling all genetic elements and their products;

ii) performing affinity selection for said tag; and

iii) isolating nucleic acid bound in step (ii) to identify a genetic element encoding a polypeptide that binds said ligand.

13. The method of claim 12, wherein step (iii) comprises amplifying said nucleic acid bound in step (ii).

14. The method of claim 11, wherein said plurality of genetic elements is a library of genetic elements.

15. The method of claim 11, further comprising the steps of iteratively repeating steps (a)-(b).

16. The method of claim 1, wherein said plurality of genetic elements are physically associated with a substrate for a reaction in which an expressed product participates, wherein a product of said reaction remains physically associated with a genetic element, and said step of separating comprises separating a genetic element that is physically associated with said product of said reaction from those that are not.

17. The method of claim 16, wherein said separating comprises affinity selection for said product of said reaction.

18. The method of claim 16, wherein said separating comprises detection of a change in an optical property of said substrate upon conversion to product.

19. The method of claim 16, wherein said separating comprises fluorescence activated sorting.

20. The method of claim 16, wherein said separating comprises the steps of:

i) pooling all genetic elements and their products; and

ii) performing affinity selection for said product.

21. The method of claim 20, further comprising the step, after step (ii), of amplifying said genetic element.

22. The method of claim 16, further comprising the steps, after step (b) but before step (c), of:

i) preparing an emulsion of said genetic elements separated in step (b);

ii) incubating said emulsion under conditions sufficient to generate a change in a detectable property of a droplet in said emulsion, wherein said change permits the identification of a genetic element encoding an expressed product.

23. The method of claim 16, wherein said plurality of genetic elements is a library.