In vitro sorting method

• US 7,638,276 B2
• Filed: 06/04/2004
• Issued: 12/29/2009
• Est. Priority Date: 07/07/1997
• Status: Expired due to Fees

• Current Assignee: UK Research and Innovation
• Original Assignee: 454 Life Sciences Corporation (Roche Holding AG)
• Inventors: Griffiths, Andrew, Tawfik, Dan
• Primary Examiner(s): Kelly; Robert M.
• Assistant Examiner(s): HIRIYANNA, KELAGINAMANE T
• Application Number:
  US10/861,930
• Publication Number:
  US 20050042648A1
• Time in Patent Office: 2,034 Days
• Field of Search: None
• US Class Current: 435/6.16
• 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 increasing the concentration of a nucleic acid molecule by polymerase chain reaction amplification comprising the steps of:

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

(b) amplifying the nucleic acid molecule in the microcapsules to form amplified copies of the nucleic acid molecule which bind to said bead; and

(c) breaking the microcapsules and selecting for said amplified copies of the nucleic acid molecule using a tag, wherein the tag comprises one member of a binding pair.

2. The method of claim 1 wherein the emulsion includes at least one emulsion stabilizer.

3. The method of claim 2 wherein the emulsion stabilizer is a non-ionic surfactant.

4. The method of claim 3 wherein the emulsion stabilizer is selected from the group consisting of sorbitan monooleate and polyoxyethylenesorbitan monooleate.

5. The method of claim 2 wherein the emulsion stabilizer is an anionic surfactant.

6. The method of claim 5 wherein the emulsion stabilizer is selected from the group consisting of sodium cholate, sodium glycocholate, sodium taurocholate, and sodium deoxycholate.

7. The method of claim 1 wherein the nucleic acid molecule comprises a biotin tag.

8. The method of claim 1 wherein the bead is selected from the group consisting of polystyrene and magnetic beads.

9. The method of claim 1 wherein a plurality of microcapsules when formed contains on average one or less than one nucleic acid molecule per microcapsule.

10. The method of claim 1 wherein a plurality of microcapsules when formed each contains on average between 5 and 1000 nucleic acid molecules.

11. The method of claim 1 wherein the binding pair is biotin -avidin/streptavidin.