Optical sorting method

• US 20050037392A1
• Filed: 06/11/2004
• Published: 02/17/2005
• Est. Priority Date: 01/07/1999
• Status: Abandoned Application

• Current Assignee: Medical Research Council (Government of United Kingdom)
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Sepp, Armin, Griffiths, Andrew, Tawfik, Dan
• Application Number:
  US10/866,237
• Publication Number:
  US 20050037392A1
• Time in Patent Office: Days
• US Class Current: 435/6
• 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: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules

Abstract

The invention describes a method for isolating one or more genetic elements encoding a gene product having a desired activity, comprising 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 the desired activity using a change in the optical properties of the genetic elements. The invention enables the in vitro evolution of nucleic acids and proteins 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 microcapsules include a nucleic acid molecule, a bead capable of being linked to the nucleic acid molecule, and an aqueous solution comprising components necessary to perform nucleic acid amplification;

(b) amplifying the nucleic acid molecule in the microcapsules to form amplified product copies of the nucleic acid molecule; and

(c) capturing the amplified product copies to the bead in the microcapsules, thereby increasing the concentration of the nucleic acid molecule.

3. 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.

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

5. The method of claim 2, wherein the water-in-oil emulsion includes at least one emulsion stabilizer.

6. The method of claim 5, wherein the emulsion stabilizer is a non-ionic surfactant.

7. The method of claim 6, wherein the emulsion stabilizer is selected from the group consisting of sorbitan monooleate and polyoxyethylenesorbitan monooleate.

8. The method of claim 5, wherein the emulsion stabilizer is an anionic surfactant.

9. The method of claim 8, wherein the emulsion stabilizer is selected from the group consisting of sodium cholate, sodium taurocholate, and sodium deoxycholate.

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

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

12. The method of claim 2 wherein the solid-phase support is a bead.

13. The method of claim 12, wherein the bead comprises a coating selected from the group consisting of avidin or streptavidin.

14. The method of claim 12, wherein the bead is selected from the group consisting of polystyrene, paramagnetic and magnetic beads.

15. 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.

16. The method of claim 2 wherein the nucleic acid molecule is genomic DNA or cDNA.

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.