In vitro sorting method
View Patent ↗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.
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.