Vitro sorting method

• US 20050042648A1
• Filed: 06/04/2004
• Published: 02/24/2005
• 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 7,638,276 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. (canceled).

2. A method for increasing concentration of a nucleic acid molecule comprising the steps of:

(a) forming aqueous microcapsules from a water-in-oil emulsion, wherein a plurality of the microcapsules include a nucleic acid molecule, and an aqueous solution comprising components necessary for nucleic acid amplification;

(b) amplifying the nucleic acid molecule in the microcapsules to form amplified copies of the nucleic acid molecule.

3. The method of claim 2 wherein the nucleic acid molecule is linked to a solid-phase support.

4. The method of claim 2 wherein the nucleic acid amplification is performed using a method selected from the group consisting of Qb replicase amplification, ligase chain reaction, self sustained sequence replication, and strand displacement amplification.

5. The method of claim 2 wherein the nucleic acid amplification is performed using polymerase chain reaction.

6. The method of claim 2 wherein the emulsion includes at least one emulsion stabilizer.

7. The method of claim 6 wherein the emulsion stabilizer is a non-ionic surfactant.

8. The method of claim 7 wherein the emulsion stabilizer is selected from the group consisting of sorbitan monooleate and polyoxyethylenesorbitan monooleate.

9. The method of claim 6 wherein the emulsion stabilizer is an anionic surfactant.

10. The method of claim 9 wherein the emulsion stabilizer is selected from the group consisting of sodium cholate, sodium glycocholate, sodium taurocholate, and sodium deoxycholate.

11. The method of claim 2 or claim 4, wherein the emulsion is thermostable.

12. The method of claim 2, wherein the nucleic acid molecule comprises a biotin tag.

13. The method of claim 3 wherein the solid-phase support is a bead.

14. The method of claim 13 wherein the bead comprises a coating selected from the group consisting of avidin and streptavidin.

15. The method of claim 13 wherein the bead is selected from the group consisting of polystyrene and magnetic beads.

16. The method of claim 2 wherein the solid-phase support is a bead, the nucleic acid amplification is performed using polymerase chain reaction, and the emulsion is thermostable.

17. The method of claim 2 wherein a plurality of microcapsules when formed each contains on average one or less than one nucleic acid molecule.

18. The method of claim 2 wherein a plurality of microcapsules when formed each contains on average between 5 and 1000 nucleic acid molecules.